The M 1 -muscarinic acetylcholine receptor subtype may play a role in learning and memory performance in the hippocampus of neonatal monosodium glutamate-obese rats.

Here, we investigate the effects of obesity induced by monosodium glutamate (MSG) on cognitive impairment and whether this model induces any alteration in the affinity, density, and subtypes of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Healthy rats were used as controls, and MSG-obese rats were selected via the Lee index > 0.300. The effects of MSG-induced obesity on hippocampal spatial learning and memory processes were evaluated by using the working memory versions of the Morris' water maze task and the evaluation of mAChRs by binding assay and their subtypes by immunoprecipitation assays. [ 3 H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (K D ) did not differ between control and MSG, indicating that affinity is not affected by obesity induced by MSG. The maximum number of binding sites (B max ) obtained in MSG subjects was lower than that obtained from control rats, indicating a decrease in the expression of total mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M 1 subtype of MSG when compared with control rats (M 2 to M 5 subtypes did not differ between control and MSG). We also observed that MSG promotes a disruption of the spatial working memory which was accompanied by a decrease in the M 1 mAChR subtype in rat hippocampus, thus suggesting deleterious long-term effects besides the obesity. In conclusion, these findings provide new insights into how obesity can influence spatial learning and memory that is hippocampal-dependent. The data suggest that the M 1 mAChR subtype protein expression is a potential therapeutic target.

The M1-muscarinic acetylcholine receptor subtype may play a role in learning and memory performance in the hippocampus of neonatal monosodium glutamate-obese rats

Here, we investigate the effects of obesity induced by monosodium glutamate (MSG) on cognitive impairment and whether this model induces any alteration in the affinity, density, and subtypes of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Healthy rats were used as controls, and MSG-obese rats were selected via the Lee index > 0.300. The effects of MSG-induced obesity on hippocampal spatial learning and memory processes were evaluated by using the working memory versions of the Morris’ water maze task and the evaluation of mAChRs by binding assay and their subtypes by immunoprecipitation assays. [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ between control and MSG, indicating that affinity is not affected by obesity induced by MSG. The maximum number of binding sites (Bmax) obtained in MSG subjects was lower than that obtained from control rats, indicating a decrease in the expression of total mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 subtype of MSG when compared with control rats (M2 to M5 subtypes did not differ between control and MSG). We also observed that MSG promotes a disruption of the spatial working memory which was accompanied by a decrease in the M1 mAChR subtype in rat hippocampus, thus suggesting deleterious long-term effects besides the obesity. In conclusion, these findings provide new insights into how obesity can influence spatial learning and memory that is hippocampal-dependent. The data suggest that the M1 mAChR subtype protein expression is a potential therapeutic target.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus.

BACKGROUND: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). METHODS: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. RESULTS: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. CONCLUSION: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.(AU)

Effects of Mlx-8, a phospholipase A2 from Brazilian coralsnake Micrurus lemniscatus venom, on muscarinic acetylcholine receptors in rat hippocampus

Background: Here, we described the presence of a neurotoxin with phospholipase A2 activity isolated from Micrurus lemniscatus venom (Mlx-8) with affinity for muscarinic acetylcholine receptors (mAChRs). Methods: The purification, molecular mass determination, partial amino acid sequencing, phospholipase A2 activity determination, inhibition of the binding of the selective muscarinic ligand [3H]QNB and inhibition of the total [3H]inositol phosphate accumulation in rat hippocampus of the Mlx-8 were determined. Results: Thirty-one fractions were collected from HPLC chromatography, and the Mlx-8 toxin was used in this work. The molecular mass of Mlx-8 is 13.628 Da. Edman degradation yielded the following sequence: NLYQFKNMIQCTNTRSWL-DFADYG-CYCGRGGSGT. The Mlx-8 had phospholipase A2 enzymatic activity. The pKi values were determined for Mlx-8 toxin and the M1 selective muscarinic antagonist pirenzepine in hippocampus membranes via [3H]QNB competition binding assays. The pKi values obtained from the analysis of Mlx-8 and pirenzepine displacement curves were 7.32 ± 0.15, n = 4 and 5.84 ± 0.18, n = 4, respectively. These results indicate that Mlx-8 has affinity for mAChRs. There was no effect on the inhibition ability of the [3H]QNB binding in hippocampus membranes when 1 µM Mlx-8 was incubated with 200 µM DEDA, an inhibitor of phospholipase A2. This suggests that the inhibition of the phospholipase A2 activity of the venom did not alter its ability to bind to displace [3H]QNB binding. In addition, the Mlx-8 toxin caused a blockade of 43.31 ± 8.86%, n = 3 and 97.42 ± 2.02%, n = 3 for 0.1 and 1 µM Mlx-8, respectively, on the total [3H]inositol phosphate content induced by 10 µM carbachol. This suggests that Mlx-8 inhibits the intracellular signaling pathway linked to activation of mAChRs in hippocampus. Conclusion: The results of the present work show, for the first time, that muscarinic receptors are also affected by the Mlx-8 toxin, a muscarinic ligand with phospholipase A2 characteristics, obtained from the venom of the Elapidae snake Micrurus lemniscatus, since this toxin was able to compete with muscarinic ligand [3H]QNB in hippocampus of rats. In addition, Mlx-8 also blocked the accumulation of total [3H]inositol phosphate induced by muscarinic agonist carbachol. Thus, Mlx-8 may be a new pharmacological tool for examining muscarinic cholinergic function.

Effects of obesity induced by high-calorie diet and its treatment with exenatide on muscarinic acetylcholine receptors in rat hippocampus.

Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO + E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO + E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO + E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO + E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO + E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO + E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO + E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.

Effects of obesity induced by high-calorie diet and its treatment with exenatide on muscarinic acetylcholine receptors in rat hippocampus

Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.

Effects of obesity induced by high-calorie diet and its treatment with exenatide on muscarinic acetylcholine receptors in rat hippocampus

Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.Here, we described the effects of obesity induced by high-calorie diet and its treatment with exenatide, an anti-diabetogenic and potential anti-obesogenic drug derived from the venom of the Gila monster Heloderma suspectum, on the affinity, density, subtypes and intracellular signaling pathways linked to activation of muscarinic acetylcholine receptors (mAChRs) in rat hippocampus. Male Wistar rats were divided into three groups: control (CT), obese induced by high-calorie diet (DIO) and DIO treated with exenatide (DIO+E). [3H]Quinuclidinyl benzilate specific binding analysis showed that the equilibrium dissociation constant (KD) did not differ among CT, DIO and DIO+E, indicating that affinity is not affected by high-calorie diet or its treatment with exenatide. On the other hand, the density of mAChRs obtained in DIO animals was lower than that obtained from CT rats, and that DIO+E restored the density of mAChRs. Immunoprecipitation assays reveal a decrease in the expression of M1 and M3 subtypes of DIO animals when compared with CT. Treatment with exenatide (DIO+E) restored the expression of the two subtypes similar to obtained from CT. On the other hand, the M2, M4 and M5 mAChR subtypes expression did not differ among CT, DIO and DIO+E. Carbacol caused a concentration-dependent increase in the accumulation of total [3H] inositol phosphate in CT, DIO and DIO+E. However, the magnitude of the maximal response to carbachol was lower in DIO when compared with those obtained from CT and DIO+E animals, which did not differ from each other. Our results indicate that obesity induced by high-calorie diet strongly influences the expression and intracellular signaling coupled to M1-M3 mAChR subtypes. The exenatide ameliorated these effects, suggesting an important role on hippocampal muscarinic cholinergic system. This action of obesity induced by high-calorie diet and its treatment with exenatide might be a key step mediating cellular events important for learning and memory.

Anti-obesogenic and hypolipidemic effects of a glucagon-like peptide-1 receptor agonist derived from the saliva of the Gila monster.

INTRODUCTION: Glucagon-like peptide-1 (GLP-1) receptor (R) agonists are a class of incretin mimetic drugs that have been used for the treatment of type 2 diabetes mellitus and also considered strong candidates for the treatment of obesity. The original prototypical drug in this class is the exenatide, a synthetic peptide with the same structure as the native molecule, exendin-4, found in the saliva of the Gila monster (Heloderma suspectum suspectum lizard). OBJECTIVES: To identify and compare the anti-obesogenic, antidyslipidemic and antidiabetogenic effects of agonism in GLP-1R by exenatide on two distinct models of obesity: induced by hypothalamic injury (MSG) or high-calorie diet (DIO). METHODS: To obtain MSG, neonatal rats were daily subcutaneously injected with 4 g monosodium glutamate/kg, for 10 consecutive days. To obtain DIO, 72-75 days old rats received hyperlipid food and 30% sucrose for drinking up to 142-145 days old. Untreated healthy rats with the same age were used as control. General biometric and metabolic parameters were measured. RESULTS: MSG was characterized by decreased naso-anal length, food and fluid intake, plasma protein and glucose decay rate per minute after insulin administration (KITT), as well as increased Lee index (body mass0.33/naso-anal length), mass of retroperitoneal and periepididymal fat pads, glycemia, triglycerides (TG), LDL and VLDL. Exenatide ameliorated KITT and food and fluid intake, and it also restored glycemia in MSG. DIO was characterized by glucose intolerance, increased body mass, Lee index, fluid intake, mass of retroperitoneal and periepididymal fat pads, glycemia, glycated hemoglobin (HbA1c), TG, VLDL and total cholesterol, as well as decreased food intake and KITT. Exenatide restored glycemia, HbA1c, TG, VLDL, total cholesterol and body mass, and it also ameliorated food and fluid intake, KITT and mass of retroperitoneal fat pad in DIO. CONCLUSIONS: The hypothalamic injury and the high-calorie diet induce dyslipidemia and glycemic dysregulation in addition to obesity in rats. The usual therapeutic dose of exenatide in humans is antidiabetogenic in both these obesity models, but is anti-obesogenic and hypolipidemic only in diet-induced obesity. Agonists of GLP-1R are promising anti-obesogenic and antidyslipidemic drugs in the early stages of the obesity, in which the integrity of the nervous system was unaffected.

Anti-obesogenic and hypolipidemic effects of a glucagon-like peptide-1 receptor agonist derived from the saliva of the Gila monster

Introduction: Glucagon-like peptide-1(GLP-1) receptor (R) agonists are a class of incretin mimetic drugs that have been used for the treatment of type 2 diabetes mellitus and also considered strong candidates for the treatment of obesity. The original prototypical drug in this class is the exenatide, a synthetic peptide with the same structure as the native molecule, exendin-4, found in the saliva of the Gila monster (Heloderma suspectum suspectum lizard). Objectives: To identify and compare the anti-obesogenic, antidyslipidemic and antidiabetogenic effects of agonism in GLP-1R by exenatide on two distinct models of obesity: induced by hypothalamic injury (MSG) or high-calorie diet (DIO). Methods: To obtain MSG, neonatal rats were daily subcutaneously injected with 4 g monosodium glutamate/kg, for 10 consecutive days. To obtain DID, 72-75 days old rats received hyperlipid food and 30% sucrose for drinking up to 142-145 days old. Untreated healthy rats with the same age were used as control. General biometric and metabolic parameters were measured. Results: MSG was characterized by decreased naso-anal length, food and fluid intake, plasma protein and glucose decay rate per minute after insulin administration (K-ITT), as well as increased Lee index (body mass(0.33)/naso-anal length), mass of retroperitoneal and periepididymal fat pads, glycemia, triglycerides (TG), LDL and VLDL. Exenatide ameliorated K-ITT and food and fluid intake, and it also restored glycemia in MSG. DIO was characterized by glucose intolerance, increased body mass, Lee index, fluid intake, mass of retroperitoneal and periepididymal fat pads, glycemia, glycated hemoglobin (HbAlc), TG, VLDL and total cholesterol, as well as decreased food intake and K-ITT. Exenatide restored glycemia, HbA1c, TG, VLDL, total cholesterol and body mass, and it also ameliorated food and fluid intake, K-ITT and mass of retroperitoneal fat pad in DIO. Conclusions: The hypothalamic injury and the high-calorie diet induce dyslipidemia and glycemic dysregulation in addition to obesity in rats. The usual therapeutic dose of exenatide in humans is anti-diabetogenic in both these obesity models, but is anti-obesogenic and hypolipidemic only in diet-induced obesity. Agonists of GLP-1R are promising anti-obesogenic and antidyslipidemic drugs in the early stages of the obesity, in which the integrity of the nervous system was unaffected.

M1 and M3 muscarinic receptors may play a role in the neurotoxicity of anhydroecgonine methyl ester, a cocaine pyrolysis product.

The smoke of crack cocaine contains cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). AEME possesses greater neurotoxic potential than cocaine and an additive effect when they are combined. Since atropine prevented AEME-induced neurotoxicity, it has been suggested that its toxic effects may involve the muscarinic cholinergic receptors (mAChRs). Our aim is to understand the interaction between AEME and mAChRs and how it can lead to neuronal death. Using a rat primary hippocampal cell culture, AEME was shown to cause a concentration-dependent increase on both total [(3)H]inositol phosphate and intracellular calcium, and to induce DNA fragmentation after 24 hours of exposure, in line with the activation of caspase-3 previously shown. Additionally, we assessed AEME activity at rat mAChR subtypes 1-5 heterologously expressed in Chinese Hamster Ovary cells. l-[N-methyl-(3)H]scopolamine competition binding showed a preference of AEME for the M2 subtype; calcium mobilization tests revealed partial agonist effects at M1 and M3 and antagonist activity at the remaining subtypes. The selective M1 and M3 antagonists and the phospholipase C inhibitor, were able to prevent AEME-induced neurotoxicity, suggesting that the toxicity is due to the partial agonist effect at M1 and M3 mAChRs, leading to DNA fragmentation and neuronal death by apoptosis.

Ovariectomy and 17ß-estradiol replacement play a role on the expression of Endonuclease-G and phosphorylated cyclic AMP response element-binding (CREB) protein in hippocampus.

The aim of the present study was to investigate the effects of different periods of ovariectomy and 17ß-estradiol (E2) replacement on the expression of Cytochrome C, apoptosis inducing factor (AIF) and Endonuclease-G (Endo-G) in mitochondrial and cytosolic fractions obtained from hippocampus of the adult female rats. In addition, the expression of phosphorylated CREB (phospho-CREB) was also analyzed in hippocampus. Ovariectomy or E2 treatment did not change the expression of Cytochrome C and AIF. Ovariectomy (15, 21 and 36 days) decreased the expression of Endo-G in the mitochondrial fractions and increased it in the cytosolic fractions obtained from hippocampus. The treatment with E2 after 15 days of ovariectomy for 7 days or 21 days, and throughout the post-ovariectomy period prevented the effects of ovariectomy on Endo-G expression. Our results suggest that ovariectomy-induced apoptotic cell death in hippocampal tissue could be mediated by Endo-G, but not by AIF, via a caspase-independent apoptotic pathway. Furthermore, ovariectomy decreased the expression of phospho-CREB and the treatment with E2 prevented these effects. In conclusion, E2 may help maintain long-term neuronal viability by regulating the expression of members of the Bcl-2 family. Regulation of Endo-G released from mitochondria, but not of Cytochrome C and AIF, is also involved in the neuroprotective actions of E2. Furthermore, CREB may be involved in the expression of Bcl-2. These data provide new understanding into the mechanisms involved in the neuroprotective role of estrogen.

Neurotoxicity of anhydroecgonine methyl ester, a crack cocaine pyrolysis product.

Smoking crack cocaine involves the inhalation of cocaine and its pyrolysis product, anhydroecgonine methyl ester (AEME). Although there is evidence that cocaine is neurotoxic, the neurotoxicity of AEME has never been evaluated. AEME seems to have cholinergic agonist properties in the cardiovascular system; however, there are no reports on its effects in the central nervous system. The aim of this study was to investigate the neurotoxicity of AEME and its possible cholinergic effects in rat primary hippocampal cell cultures that were exposed to different concentrations of AEME, cocaine, and a cocaine-AEME combination. We also evaluated the involvement of muscarinic cholinergic receptors in the neuronal death induced by these treatments using concomitant incubation of the cells with atropine. Neuronal injury was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and lactate dehydrogenase (LDH) assays. The results of the viability assays showed that AEME is a neurotoxic agent that has greater neurotoxic potential than cocaine after 24 and 48 h of exposure. We also showed that incubation for 48 h with a combination of both compounds in equipotent concentrations had an additive neurotoxic effect. Although both substances decreased cell viability in the MTT assay, only cocaine increased LDH release. Caspase-3 activity was increased after 3 and 6 h of incubation with 1mM cocaine and after 6 h of 0.1 and 1.0mM AEME exposure. Atropine prevented the AEME-induced neurotoxicity, which suggests that muscarinic cholinergic receptors are involved in AEME's effects. In addition, binding experiments confirmed that AEME has an affinity for muscarinic cholinergic receptors. Nevertheless, atropine was not able to prevent the neurotoxicity produced by cocaine and the cocaine-AEME combination, suggesting that these treatments activated other neuronal death pathways. Our results suggest a higher risk for neurotoxicity after smoking crack cocaine than after cocaine use alone.

Characterization of a new muscarinic toxin from the venom of the Brazilian coral snake Micrurus lemniscatus in rat hippocampus

We have isolated a new muscarinic protein (MT-Mlá) from the venom of the Brazilian coral snake Micrurus lemniscatus.The MT-Mlá was able to displace the [3H]QNB binding in the hippocampus of rats. The bindingcurve in competition experiments with MT-Mlá was indicative of two types of [3H]QNB-binding site with pKivalues of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor(mAChR) subtypes may be the target proteins of MT-Mlá. The MT-Mlá and the M1 antagonist pirenzepinecaused a dose-dependent block on total [3H]inositol phosphate accumulation induced by carbachol. TheIC50 values for MT-Mlá and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlá has antagonist effect on mAChRs in rat hippocampus.The results of the present study show, for the first time, that mAChRs function is drasticallyaffected by MT-Mlá since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs.

Characterization of a new muscarinic toxin from the venom of the Brazilian coral snake Micrurus lemniscatus in rat hippocampus.

AIMS: We have isolated a new muscarinic protein (MT-Mlα) from the venom of the Brazilian coral snake Micrurus lemniscatus. MAIN METHODS: This small protein, which had a molecular mass of 7,048Da, shared high sequence homology with three-finger proteins that act on cholinergic receptors. The first 12 amino acid residues of the N-terminal sequence were determined to be: Leu-Ile-Cys-Phe-Ile-Cys-Phe-Ser-Pro-Thr-Ala-His. KEY FINDINGS: The MT-Mlα was able to displace the [(3)H]QNB binding in the hippocampus of rats. The binding curve in competition experiments with MT-Mlα was indicative of two types of [(3)H]QNB-binding site with pK(i) values of 9.08±0.67 and 6.17±0.19, n=4, suggesting that various muscarinic acetylcholine receptor (mAChR) subtypes may be the target proteins of MT-Mlα. The MT-Mlα and the M(1) antagonist pirenzepine caused a dose-dependent block on total [(3)H]inositol phosphate accumulation induced by carbachol. The IC(50) values for MT-Mlα and pirenzepine were, respectively, 33.1 and 2.26 nM. Taken together, these studies indicate that the MT-Mlα has antagonist effect on mAChRs in rat hippocampus. SIGNIFICANCE: The results of the present study show, for the first time, that mAChRs function is drastically affected by MT-Mlα since it not only has affinity for mAChRs but also has the ability to inhibit mAChRs.