|Year : 2016 | Volume
| Issue : 2 | Page : 115-120
Evaluate the effect of bethanechol on blood glucose levels in euglycemic Wistar albino rats through oral glucose tolerance test
RN Suresha, Siddamma Amoghimath, MK Jayanthi, SL Shruthi
Department of Pharmacology, JSS Medical College, Mysore, Karnataka, India
|Date of Web Publication||30-Jun-2016|
JSS Medical College, Mysore, Karnataka
Source of Support: None, Conflict of Interest: None
Objective: To evaluate the effect of bethanechol on blood glucose levels in euglycemic Wistar albino rats through oral glucose tolerance test (OGTT). Materials and Methods: Twelve Wistar albino rats weighing around 150-200 g of either sex were randomly selected from the central animal facility, and were divided into two groups. The control group received distilled water (25 mL/kg body wt.) per orally, test groups received bethanechol (3.6 mg/kg/day) per orally for 5 days. On the fifth day, following overnight fasting, 1 h after drug administration in all the groups of rats OGTT was performed, by administering oral glucose in dose of 0.6 gm/kg body weight. The capillary blood glucose (CBG) levels were measured at 0 min, 60 min, and 150 min, by rat tail snipping method using glucometer (ACCUCHEK). Results: The CBG levels of the bethanechol group was less when compared to the control group at all time intervals and the difference was statistically significant. Conclusion: Bethanechol showed the hypoglycemic activity when given for 5 days orally to euglycemic albino rats through OGTT.
Keywords: Bethanechol, capillary blood glucose (CBG), diabetes, euglycemic, oral glucose tolerance test (OGTT)
|How to cite this article:|
Suresha R N, Amoghimath S, Jayanthi M K, Shruthi S L. Evaluate the effect of bethanechol on blood glucose levels in euglycemic Wistar albino rats through oral glucose tolerance test. Muller J Med Sci Res 2016;7:115-20
|How to cite this URL:|
Suresha R N, Amoghimath S, Jayanthi M K, Shruthi S L. Evaluate the effect of bethanechol on blood glucose levels in euglycemic Wistar albino rats through oral glucose tolerance test. Muller J Med Sci Res [serial online] 2016 [cited 2022 Aug 16];7:115-20. Available from: https://www.mjmsr.net/text.asp?2016/7/2/115/185011
| Introduction|| |
Diabetes mellitus (DM) consists of a group of syndromes characterized by hyperglycemia, altered metabolism of lipids, carbohydrates, and proteins and an increased risk of complications that include vascular and neurological abnormalities. 
Type II DM is at present one of the most challenging health-care problems, which requires optimum management. At present, the treatment of DM includes insulin, sulfonylureas, biguanides, α-glucosidase inhibitors, dipeptidyl peptidase (DPP)-4 inhibitors, thiazolidinediones, glucagon-like peptide (GLP)-1 receptor agonists, amylin agonists, medical nutrition therapy, and lifestyle modification.
Glucose gets phosphorylated by glucokinase to glucose 6 phosphate, which enters the glycolytic pathway, producing nicotinamide adenine dinucleotide phosphate (NADPH) and increased ratio of adenosine triphosphate/adenosine diphosphate (ATP/ADP). Elevated ATP/ADP ratio inhibits ATP-sensitive K + (KATP) channel leading to depolarization. The KATP channel consists of (kir6.2) inward rectifying channel and a closely related sulfonylurea receptor (SUR). The closure of K-ATP channel depolarizes the cell membrane to potentials above -55 mV. Now this leads to the activation of T-type (at voltages above -60 mV) and L-type Ca 2+ channels (above -50 mV) initiates the action potential. During the upstroke of the action potential, voltage-gated Na + channels also open (above -40 mV), leading to a further acceleration of the upstroke and sufﬁcient depolarization to activate P/Q-type Ca 2+ channels (above -20 mV). Ca 2+ inﬂux via P/Q-type (and to a lesser extent L-type) directly triggers exocytosis of insulin granules. Na + current is important for glucose-induced insulin secretion. Hence, it signifies that sodium channels are important for action potential generation and insulin secretion.
Voltage-gated plasmalemmal ion channels play a fundamental role in stimulus secretion coupling in cells, and Ca 2+ inﬂux through voltage-gated Ca 2+ channels triggers exocytosis of insulin containing secretory granules. Voltage-gated Ca 2+ channels are activated by coordinated ﬂuctuations of the cell membrane potential (electrical activity), which are initiated by the glucose-induced closure of KATP channels and dependent on voltage-gated Na + and K + channels. 
Oral glucose tolerance test
The oral glucose tolerance test (OGTT) is a measure of the glucose-induced insulin secretion and its mediated glycemic changes. This study used OGTT for normoglycemic rats with some modifications to the standard method (Duvigneaud and Karr, 1925) to assess the effect of bethanechol on glucose-induced glycemic alteration. 
Bethanechol is a muscarinic receptor (M 3 ) agonist. It is used in the treatment of urinary retention; inadequate emptying of the bladder when organic obstruction is absent; diabetic autonomic neuropathy; and certain cases of chronic hypotonic, myogenic, and neurogenic bladder. In gastro intestinal tract (GIT), bethanechol stimulates peristalsis, increased motility, and increased lower esophageal sphincter pressure. Bethanechol has a plasma half-life of 2 h and is eliminated through kidney. 
The acetylcholine/vagus effects on pancreatic insulin release are mediated by the activation of muscarinic acetylcholine receptors located on the pancreatic β-cells.
M3 receptors are present in visceral smooth muscles, iris, ciliary muscle, exocrine glands, endocrine glands, and vascular endothelium. 
M3 receptors are G q -protein coupled and activate the membrane bound phospholipase C (PLC) generating inositol triphosphate (IP3) and diacylglycerol (DAG) which in turn release Ca 2+ intracellularly causing depolarization.
Cholinomimetic drugs produce actions similar to that of acetylcholine, either by directly interacting with cholinergic receptors (Cholinergic agonist) or by increasing availability of acetylcholine at these sites (anticholinesterase). 
Depending upon the site of action the peripheral actions are mediated.
At the β-cell level, acetylcholine binds to M3 receptors and activates several transduction pathways; one of the major pathways is PLC, which mainly generates IP3 and DAG, a potent phosphokinase C (PKC) activator. DAG is liposoluble that remains in the plasma membrane to which it causes translocation of its target, protein kinases-C. This translocation also requires Ca 2+ , and phosphatidylserine activates the kinases, which can then phosphorylate proteins. Thus, acetylcholine potentiates insulin secretion by amplifying the action of the triggering signal. 
IP3 which is hydrosoluble, diffuses into the cytoplasm and binds to receptors present on the membranes of endoplasmic reticulum, causing active pumping by Ca 2+ Atp-ase (SERCA pump). This results in the opening of channels through which the Ca 2+ diffuses from the organelle to the cytoplasm. The consequences is an increase in Ca 2+ that usually displays two phases, an initial large peak followed by a smaller sustained elevation of insulin. The amplitude and pattern of the Ca 2+ change are very much dependent on the ambient glucose concentration.
The sequential events are depicted in [Figure 1].
|Figure 1: The figure shows the schematic representation of mechanism of action of insulin release through muscarinic receptors (M3) on pancreatic β-cell M3 = muscarinic M3 receptors, PLC = phospholipase C, PIP2 = phosphatidylinositol 4, 5-bisphosphate, IP3 = inositol triphosphate, ER = endoplasmic reticulum, DAG = diacylglycerol, PKC = phosphokinase C|
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The conditions, such as intestinal hypomotility syndrome, gastroparesis, detrusor hypotonic, and neurogenic bladder, are established manifestations of DM. 
Bethanechol that is given in the treatment of above conditions acts as muscarinic agonist, which mediates smooth muscle contraction in both the tissues. Hence, bethanechol is helpful in the treatment of diabetic gastrointestinal hypomotility condition by increasing peristalsis, increased motility and also simultaneously helps to maintain normal blood glucose levels through activation of PLC, which potentiates insulin secretion by increasing the triggering signal for insulin secretion (via IP3) and amplifying insulin action (via DAG).
Thus, the mechanisms by which cholinomimetics acts to cause insulin secretion are as follows:
- Major pathways are activation of PLC, which mainly generates IP3 and DAG, a potent PKC activator. ,,
- Acetylcholine also depolarizes the plasma membrane of β-cells by a Na + or nonspecific cationic-dependent mechanism.
Acetylcholine acting through M3 receptors, by activation of phospholipase C, generates IP3 and DAG and also depolarizes the membrane of insulin stored granules by sodium channel and causes secretion of insulin and therefore decreases the blood glucose level. Bethanechol is a cholinergic agonist, which is hypothesized to exhibit the same activity.
| Materials and Methods|| |
The study was conducted after obtaining the approval of Institution Animal Ethical Committee (IAEC).
Wistar albino rats of either sex of average weight 150-200 gm (aged 3-4 months) were used in the experiments. The albino rats were bred in Central Animal Facility. The study was done in the Department of Pharmacology during the month of March 2014. Animals were acclimatized to the laboratory conditions for at least 1 h before testing, and then they were used during experiments. The doses of drugs were based on the human daily dose converted to that of animal dose using the following standard formula: . 
Drugs and chemicals
Tab. bethanechol (25 mg) (Ciplapharmaceuticals, India) was dissolved in distilled water and immediately administered orally, distilled water was given orally, and 0.6 mg/kg of glucose mixed in distilled water was also given for the OGTT.
The Wistar albino rats were divided into two groups with six animals (n = 6) in each group (control and test groups). The test drug bethanechol (3.6 mg/kg/day) and distilled water (25 mL/kg/day) were administered orally for 5 days.
Group 1: Distilled water-25 mL/kg/day (orally)
Group 2: Bethanechol-3.6 mg/kg/day (orally)
All rats were fasted overnight before the fifth day. On the fifth day, 1 h after the last dose of the respective drug, OGTT was performed. All the rats were given glucose (0.6 gm/kg body weight) orally using gavage tube. Following this, the capillary blood glucose (CBG) (obtained by tail snipping) was assessed at 0 min, 60 min, and 150 min of time intervals using a glucometer (ACCUCHEK from Roche Diagnostics).
The results were an analyzed by calculating the mean values, standard deviation, and using t-test and the analysis of variance (ANOVA) at different time intervals within the same group followed by an independent sample t-test between the two groups. The values were compared at 0.05 level of significance for the corresponding degrees of freedom. P < 0.05 were considered as significant. All the statistical analysis was done by using IBM SPSS 21 software (United States).
| Results|| |
The bethanechol group showed fall in CBG levels when compared to the control group at all time intervals, with maximum fall at 60 min. Thus, the fall in CBG levels of bethanecol group was statistically significant (P < 0.05) at 0 min, 60 min and 120 min respectively.
The capillary blood glucose levels of bethanecol group when compared to control group was lower at all-time intervals, the CBG leve linter-interval differences of bethanecol i.e., 0-60 min, 60-150 min and 0-150 min is lower when compared to control and is statistically significant (P < 0.05).
| Discussion|| |
In the present study, as per [Table 1] and [Table 2], the bethanechol group showed decrease in the CBG levels at all time intervals of OGTT (i.e., 0 min, 60 min, and 150 min) when compared to the control group. The CBG level of the bethanechol group at 0 min was 11.16 ± 1.71 that was more when compared to the control group (i.e., 15.25%), which indicates indirectly that bethanechol increased basal secretion of insulin. The CBG level bethanechol group at 60 min was 20 ± 0.11, which is more when compared to the control group (i.e., 19.9%), which indicates that bethanechol causes more glucose dependent insulin secretion from pancreatic β-cells. The CBG level of the bethanechol group at 150 min was 11.5 ± 0.58, which is more when compared to the control group (i.e., 14.02%) because of sustained action of bethanechol on pancreatic β-cells. The quantum of fall in CBG levels of bethanechol group at 0 min and 150 min is almost equal. [Graph 1 [Additional file 1]] showed the Capillary Blood glucose CBG levels of control and bethanecol group at different time intervals. [Graph 2 [Additional file 2]] and [Graph 3 [Additional file 3]] showed the reduction in CBG levels and fall in percentage in the CBG level of the bethanechol group compared to the control group.
|Table 1: Capillary blood glucose (CBG) levels in the control and bethanechol groups, and the difference between the control and bethanechol groups at corresponding time intervals|
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|Table 2: Percentage fall in capillary blood glucose (CBG) level in the bethanechol group when compared to the control group|
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In [Table 3], the inter-interval difference of the bethanechol group at 0-60 min is maximum, which indicates glucose dependent insulin release and inter-interval difference at 60-150 min of bethanechol group is more compared to 0-150 min because of sustained effect on pancreatic β-cell. The interval difference at 0-150 min is the total combined effect of bethanechol on pancreatic β-cells. [Graph 4 [Additional file 4]] showed the difference in the CBG levels of the bethanechol and control groups at time intervals 0 min, 60 min, and 150 min of OGTT. [Table 4] and [Graph 5 [Additional file 5]] showed difference in CBG values between control group and bethanecol group at various time intervals.
|Table 3: The difference in the CBG levels of the bethanechol and control groups at time intervals 0 min, 60 min, and 150 min of OGTT|
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|Table 4: Depicts difference in CBG values between the control group and the bethenecol group at various time intervals|
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The above findings indicate that bethanechol acts as a hypoglycemic drug in normal Wistar albino rats. OGTT is used to assess the glucose tolerance that indirectly indicates the insulin sensitivity and β-cell function. Hence, in diabetics and prediabetics it can be assumed that bethanechol causes a decrease in blood glucose levels.
| Conclusion|| |
The test drug bethanechol showed significant decrease in CBG level in euglycemic Wistar albino rats when compared to that of the control group through OGTT. The hypoglycemic activity of bethanechol was maximum during the 60 min, which justifies the hypothesis stated above and enhances the glucose dependent insulin release.
Thus, to conclude bethanechol causes decrease in blood glucose levels in euglycemic albino rats through the stimulation of muscarinic receptor and activation of phospholipase C that generates IP3 and DAG and also depolarizes membrane by sodium channel and causes secretion of insulin.
We would like to thank our institution for permitting and providing the necessary assistance for the study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4]