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Muscarinic receptors and insulin concentration in the rat pancreas after chronic alcohol intake and cholinergic stimulation
ExpToxic Patho11996; 48: 77-79 Gustav FischerVerlag lena
Short communication
Departments of Pathology'), Clinical Pharmacology" and Surgery" , University of Turku, Turku, Finland
Muscarinic receptors and insulin concentration in the rat pancreas after chronic alcohol intake and cholinergic stimulation V.1. O. LAINE'!, R. HUUPPONEN 2l, T. KAILA2), 1. GRONROOS 3) and T. J. NEVALAINENI) With 2 figures Received: January 17, 1995; Revised: May 10, 1995; Accepted: May 17, 1995 Address for correspondence: VELI J. O. LAINE, MD, Departmentof Pathology, Universityof Turku, Kiinamyllynkatu 10, 20520 Turku, Finland; Tele: 358-21-6337370, Fax: 358-21 -6337459, E-mail: [email protected] Key words: Carbachol; Ethanol, chronic intake; Insulin; Muscarinic receptors; N-methylscopolamine; Pancreas; Alcohol, chronic intake.
Chronic alcohol intake causes down-regulation of muscarinic receptors and an increase in the concentrations of acetylcholine in pancreatic parenchyma. Muscarinic stimulation has been implicated in the pathogenesis of alcoholinduced acute pancreatitis (GRONROOS et al. 1989). Insulin secretion from beta-cells is regulated by parasympathetic nerves, gastrointestinal hormones and glucose (BERTHOUD 1984). Muscarinic stimulation of the rat pancreas with carbachol induces a glucose-dependent insulin release (KONRADet al. 1992). The purpose of the current work was to study the effect of chronic alcohol intake on insulin concentration and muscarinic receptors in the rat pancreas.
mals were randomly divided into two groups. Twenty-three animals were offered alcoholas a 15 % (w/v) solution of ethanol in water for three months. Twenty-two control animals were offered water instead of alcohol. The animals were housed in individualcages and had free access to food. The alcohol intake was measured from the total 15 % w/v ethanol consumption of animals after a threemonths' test period. The daily consumption of alcohol was three grams ethanol per rat. After the three months' test period, twelve alcohol-fed and twelve control animals were exposed to cholinergic stimulation with carbachol (Sigma, 250 ug/kg of body weight i.p.), as described earlier (BILCHICK et al. 1990). The animals were decapitated four hours after the carbachol injection. A 0.8-1 gram sampleof pancreatic tissue was trimmed free of fat andprepared for immediate determination of muscarinic receptorbinding and proteincontent. Muscarinic receptors were measured by using N-(3H)-methy1scopo1amine (NMS, 73.8 Ci/mmo1, New England Nuclear) as the radioligand. Preparation of pancreatic plasma membranes, determination of NMS binding and protein determinations were performed as describedearlier (GRONROOSet al. 1989). A 0.1 gram sample from the splenic lobe of the pancreas was placed in liquid nitrogen and stored at -70 °C for the determination of insulin concentration that was performed by a radioimmunoassay (Novo Nordisk NS , Denmark) as described earlier (HUUPPONEN et al. 1992). The results are expressed as the mean ± S.E.M. Student's t-test for independent samples was used for statistical analysis. Pearson' s linear regression analysis was used to compare NMS bindingto pancreatic plasma membranes and pancreatic insulin concentration.
Material and methods
Results
Forty-five male Spraque-Dawley rats were kept on a standard laboratory diet. At the age of eight weeks, the ani-
There was a strongcorrelationbetweenthe NMS binding to pancreatic plasma membranes and pancreatic insulin
Summary The effects of chronic alcohol intake and carbachol stimulation on pancreatic muscarinic receptor binding and insulin concentrations were studied in the rat pancreas. There was a strong correlation between the number of muscarinic receptors and the concentration of insulin in the pancreas. The concentration of insulin decreased in the pancreas after long-term ethanol exposure andincreased after carbacholstimulation. These results indicate that the secretion of insulin is mediated via the muscarinic receptor pathway, and that the changes in thenumber of muscarinic receptors mayhave a role in insulin deficiency after long-term alcohol consumption.
Introduction
ExpToxic Pathol 48 (1 996) 1
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Muscarinic receptor binding DPM/mg protein
Fig. 1. The correlation between muscarinic receptor binding and insulin concentration in rat pancreas. p =0.0003, r =0.71, n
=21.
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Fig. 2. The concentration of insulin in rat pancreas after three month's alcohol intake and four hours after carbachol stimulation. Group 1: water-drinking control animals,group 2: three month's alcohol intake, group 3: water-drinking control animals + carbachol stimulation, group 4: three month's alcohol intake + carbachol stimulation. Each column represents the mean ± S.E.M. of 10-12 experiments. p = 0.050 between groups 1 and 2, p = 0.037 between groups 1 and 3, and p =0.018 between groups 2 and 4; Student's t-test for independent samples.
concentration both in the control and in the ethanol-treated groups (r = 0.75, P = 0.013 in the controls, and r = 0.62, p =0.040 in the ethanol-treated group). Figure 1 represents the correlation between the NMS-binding to pancreatic plasma membranes and pancreatic insulin concentration in twenty-one non-carbachol-treated animals examined (r = 0.71, P = 0.0003). After stimulation with carbachol, no correlation between the concentration of insulin and NMS-binding in pancreatic tissue was observed. 78
Exp Toxic Pathol 48 (1996) 1
The concentration of insulin in pancreatic tissue was lower in the alcohol-fed than in the control animals (22.0 ± 2.43 vs. 29.6 ± 2.83 ug/mg protein, p =0.05). Cholinergic stimulation increased the pancreatic insulin concentration significantly both in the alcohol-fed (p =0.02) and control (p =0.04) animals (fig. 2). Interestingly, carbachol stimulation did not increase NMS binding after chronic alcohol intake (1599 ± 212 and 1872 ± 218 dpm/mg protein in non-carbachol-treated and carbachol-treated animals, respectively, p =0.39), while the increase was significant in the water-drinking control group (from 1962 ± 236 to 2943 ± 248 dpm/mg protein, p =0.01).
Discussion Long-term alcohol exposure decreases the amount of NMS binding, but does not effect the affinity of NMS to muscarinic receptors in the pancreas (GRONROOS et al. 1989). Thus, the changes in the NMS radioligand binding reflect changes in the number of muscarinic receptors. We found previously that the cholinergic stimulation both in intact and alcohol-fed rats stimulates the release of pancreatic phospholipase A2 into the circulation and interferes with muscarinic receptor binding in the pancreas (GRONROOS et al. 1994). Insulin secretion from pancreatic beta-cells is partly mediated by muscarinic receptors (VERCHERE et al. 1991). Muscarinic stimulation increases insulin secretion via phospholipase A2 activation and subsequent accumulation of arachidonic acid in beta-cells (KONRAD et al. 1992). Calcium entry and lipid messengers, such as diacylglycerol, are most probably also involved in this process (TURK et al. 1993). Cholinergic secretory stimulation has been reported to be even more potent in pancreatic beta-cells than in exocrine pancreatic acinar cells (OTSUKI et al. 1989). Our current finding that the concentration of insulin is strongly correlated to muscarinic receptor binding in rat pancreatic tissue supports the idea that the secretion of insulin is mediated via the muscarinic receptor pathway, and that the changes in the number of muscarinic receptors may have a role in insulin deficiency after long-term alcohol consumption. In the current study, the correlation between the NMS binding in the pancreas and pancreatic insulin concentration after three months' alcohol consumption remained significant. It is hypothesised that long-term alcohol consumption alone does not affect the regulation of insulin concentration via muscarinic receptors. The possible explanation for the finding that there was no correlation between muscarinic receptor binding and insulin concentration in the pancreas after cholinergic stimulation may be due to marked differences in the insulin responses between individual test animals. This may lead to loss of correlation in the carbachol-stimulated animals. The present experimental model is consistent with altered food and water intake as consequences of chronic alcoholism and thus the changes in food and water con-
sumption may participate in mediating the effects of chronic alcohol intake on NMS-binding and insulin concentration (GRONROOS et al. 1989). It can be concluded that the concentration of insulin in rat pancreatic tissue decreases after chronic alcohol intake and increasesafter cholinergicstimulation. Moreover, the concentration of insulin in pancreatic tissue correlates well with the binding of NMS to muscarinic receptors. Whether the decrease in the pancreatic insulin concentration after chronic alcohol intake is due to down-regulation of pancreatic muscarinic receptors remains to be established. Acknowledgements: This study was supported by the Finnish Foundation for Alcohol Studies, the University of Turku Foundation, the Cancer Society of South-Western Finland, and the Academy of Finland.
References BERTHOUD HR: The relative contribution of the nervous system, hormones and metabolites to the total insulinresponse during a meal in the rat. Metabolism 1984; 33: 18-25.
BILCHICK AJ, ZUCKER KA, ADRIAN TE, MODLIN 1M: Ameliorationof cholinergic-induced pancreatitis with a selective cholecystokinin receptor antagonist. Arch Surg 1990; 125: 1546-1549. GRONROOS JM, KAlLA T, AHO HJ, NEVALAINEN TJ: Decrease in the number of muscarinic receptors in rat pancreas after chronic alcohol intake. Pharmacol Toxicol 1989;64: 356-359. GRONROOS JM, LAINE J, KAlLA T, NEVALAINEN TJ: Chronic alcohol intake and carbachol-induced acute pancreatitis in the rat. Exp Toxic Patholl994; 46: 163-167. HUUPPONEN R, SAXEN U, KouLU M: The efficacyof glibenclamidein rats with type II diabetes induced by neonatal streptozotocin. Diabetes Res 1992;21: 59-63. KONRAD RJ, JOLLY YC, MAJOR C, WOLF BA: Carbachol stimulation of phospholipase A2 and insulin secretion in pancreatic islets. Biochem J 1992; 287: 283-290. OTSUKI M, NAKAMURA T, Y. OKABAYASHI Y, et al.: Effect of a new cholinergic agonist,aclatoniumnapadisilate, on exocrine and endocrine rat pancreas. Dig Dis Sci 1989; 34: 1249-1256. TURK J, GROSS RW, RAMANADHAM S: Amplification of insulin secretion by lipidmessengers. Diabetes 1993; 42:367-374. VERCHERE CB, KWOK YN, BROWN JC: Modulation of acethylcholine-stimulated insulin release by glucose and gastric inhibitory polypeptide. Pharmacoll99l; 42: 273-282.
Exp Toxic Patho! 48 (1996) !
79
Short communication
Departments of Pathology'), Clinical Pharmacology" and Surgery" , University of Turku, Turku, Finland
Muscarinic receptors and insulin concentration in the rat pancreas after chronic alcohol intake and cholinergic stimulation V.1. O. LAINE'!, R. HUUPPONEN 2l, T. KAILA2), 1. GRONROOS 3) and T. J. NEVALAINENI) With 2 figures Received: January 17, 1995; Revised: May 10, 1995; Accepted: May 17, 1995 Address for correspondence: VELI J. O. LAINE, MD, Departmentof Pathology, Universityof Turku, Kiinamyllynkatu 10, 20520 Turku, Finland; Tele: 358-21-6337370, Fax: 358-21 -6337459, E-mail: [email protected] Key words: Carbachol; Ethanol, chronic intake; Insulin; Muscarinic receptors; N-methylscopolamine; Pancreas; Alcohol, chronic intake.
Chronic alcohol intake causes down-regulation of muscarinic receptors and an increase in the concentrations of acetylcholine in pancreatic parenchyma. Muscarinic stimulation has been implicated in the pathogenesis of alcoholinduced acute pancreatitis (GRONROOS et al. 1989). Insulin secretion from beta-cells is regulated by parasympathetic nerves, gastrointestinal hormones and glucose (BERTHOUD 1984). Muscarinic stimulation of the rat pancreas with carbachol induces a glucose-dependent insulin release (KONRADet al. 1992). The purpose of the current work was to study the effect of chronic alcohol intake on insulin concentration and muscarinic receptors in the rat pancreas.
mals were randomly divided into two groups. Twenty-three animals were offered alcoholas a 15 % (w/v) solution of ethanol in water for three months. Twenty-two control animals were offered water instead of alcohol. The animals were housed in individualcages and had free access to food. The alcohol intake was measured from the total 15 % w/v ethanol consumption of animals after a threemonths' test period. The daily consumption of alcohol was three grams ethanol per rat. After the three months' test period, twelve alcohol-fed and twelve control animals were exposed to cholinergic stimulation with carbachol (Sigma, 250 ug/kg of body weight i.p.), as described earlier (BILCHICK et al. 1990). The animals were decapitated four hours after the carbachol injection. A 0.8-1 gram sampleof pancreatic tissue was trimmed free of fat andprepared for immediate determination of muscarinic receptorbinding and proteincontent. Muscarinic receptors were measured by using N-(3H)-methy1scopo1amine (NMS, 73.8 Ci/mmo1, New England Nuclear) as the radioligand. Preparation of pancreatic plasma membranes, determination of NMS binding and protein determinations were performed as describedearlier (GRONROOSet al. 1989). A 0.1 gram sample from the splenic lobe of the pancreas was placed in liquid nitrogen and stored at -70 °C for the determination of insulin concentration that was performed by a radioimmunoassay (Novo Nordisk NS , Denmark) as described earlier (HUUPPONEN et al. 1992). The results are expressed as the mean ± S.E.M. Student's t-test for independent samples was used for statistical analysis. Pearson' s linear regression analysis was used to compare NMS bindingto pancreatic plasma membranes and pancreatic insulin concentration.
Material and methods
Results
Forty-five male Spraque-Dawley rats were kept on a standard laboratory diet. At the age of eight weeks, the ani-
There was a strongcorrelationbetweenthe NMS binding to pancreatic plasma membranes and pancreatic insulin
Summary The effects of chronic alcohol intake and carbachol stimulation on pancreatic muscarinic receptor binding and insulin concentrations were studied in the rat pancreas. There was a strong correlation between the number of muscarinic receptors and the concentration of insulin in the pancreas. The concentration of insulin decreased in the pancreas after long-term ethanol exposure andincreased after carbacholstimulation. These results indicate that the secretion of insulin is mediated via the muscarinic receptor pathway, and that the changes in thenumber of muscarinic receptors mayhave a role in insulin deficiency after long-term alcohol consumption.
Introduction
ExpToxic Pathol 48 (1 996) 1
77
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•
•
•
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.E
• II·"
0 500
1000
1500
2000
2500
3000
3500
Muscarinic receptor binding DPM/mg protein
Fig. 1. The correlation between muscarinic receptor binding and insulin concentration in rat pancreas. p =0.0003, r =0.71, n
=21.
40
c
'2
e0.
30
~
Cl
E
20
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Experimental groups
Fig. 2. The concentration of insulin in rat pancreas after three month's alcohol intake and four hours after carbachol stimulation. Group 1: water-drinking control animals,group 2: three month's alcohol intake, group 3: water-drinking control animals + carbachol stimulation, group 4: three month's alcohol intake + carbachol stimulation. Each column represents the mean ± S.E.M. of 10-12 experiments. p = 0.050 between groups 1 and 2, p = 0.037 between groups 1 and 3, and p =0.018 between groups 2 and 4; Student's t-test for independent samples.
concentration both in the control and in the ethanol-treated groups (r = 0.75, P = 0.013 in the controls, and r = 0.62, p =0.040 in the ethanol-treated group). Figure 1 represents the correlation between the NMS-binding to pancreatic plasma membranes and pancreatic insulin concentration in twenty-one non-carbachol-treated animals examined (r = 0.71, P = 0.0003). After stimulation with carbachol, no correlation between the concentration of insulin and NMS-binding in pancreatic tissue was observed. 78
Exp Toxic Pathol 48 (1996) 1
The concentration of insulin in pancreatic tissue was lower in the alcohol-fed than in the control animals (22.0 ± 2.43 vs. 29.6 ± 2.83 ug/mg protein, p =0.05). Cholinergic stimulation increased the pancreatic insulin concentration significantly both in the alcohol-fed (p =0.02) and control (p =0.04) animals (fig. 2). Interestingly, carbachol stimulation did not increase NMS binding after chronic alcohol intake (1599 ± 212 and 1872 ± 218 dpm/mg protein in non-carbachol-treated and carbachol-treated animals, respectively, p =0.39), while the increase was significant in the water-drinking control group (from 1962 ± 236 to 2943 ± 248 dpm/mg protein, p =0.01).
Discussion Long-term alcohol exposure decreases the amount of NMS binding, but does not effect the affinity of NMS to muscarinic receptors in the pancreas (GRONROOS et al. 1989). Thus, the changes in the NMS radioligand binding reflect changes in the number of muscarinic receptors. We found previously that the cholinergic stimulation both in intact and alcohol-fed rats stimulates the release of pancreatic phospholipase A2 into the circulation and interferes with muscarinic receptor binding in the pancreas (GRONROOS et al. 1994). Insulin secretion from pancreatic beta-cells is partly mediated by muscarinic receptors (VERCHERE et al. 1991). Muscarinic stimulation increases insulin secretion via phospholipase A2 activation and subsequent accumulation of arachidonic acid in beta-cells (KONRAD et al. 1992). Calcium entry and lipid messengers, such as diacylglycerol, are most probably also involved in this process (TURK et al. 1993). Cholinergic secretory stimulation has been reported to be even more potent in pancreatic beta-cells than in exocrine pancreatic acinar cells (OTSUKI et al. 1989). Our current finding that the concentration of insulin is strongly correlated to muscarinic receptor binding in rat pancreatic tissue supports the idea that the secretion of insulin is mediated via the muscarinic receptor pathway, and that the changes in the number of muscarinic receptors may have a role in insulin deficiency after long-term alcohol consumption. In the current study, the correlation between the NMS binding in the pancreas and pancreatic insulin concentration after three months' alcohol consumption remained significant. It is hypothesised that long-term alcohol consumption alone does not affect the regulation of insulin concentration via muscarinic receptors. The possible explanation for the finding that there was no correlation between muscarinic receptor binding and insulin concentration in the pancreas after cholinergic stimulation may be due to marked differences in the insulin responses between individual test animals. This may lead to loss of correlation in the carbachol-stimulated animals. The present experimental model is consistent with altered food and water intake as consequences of chronic alcoholism and thus the changes in food and water con-
sumption may participate in mediating the effects of chronic alcohol intake on NMS-binding and insulin concentration (GRONROOS et al. 1989). It can be concluded that the concentration of insulin in rat pancreatic tissue decreases after chronic alcohol intake and increasesafter cholinergicstimulation. Moreover, the concentration of insulin in pancreatic tissue correlates well with the binding of NMS to muscarinic receptors. Whether the decrease in the pancreatic insulin concentration after chronic alcohol intake is due to down-regulation of pancreatic muscarinic receptors remains to be established. Acknowledgements: This study was supported by the Finnish Foundation for Alcohol Studies, the University of Turku Foundation, the Cancer Society of South-Western Finland, and the Academy of Finland.
References BERTHOUD HR: The relative contribution of the nervous system, hormones and metabolites to the total insulinresponse during a meal in the rat. Metabolism 1984; 33: 18-25.
BILCHICK AJ, ZUCKER KA, ADRIAN TE, MODLIN 1M: Ameliorationof cholinergic-induced pancreatitis with a selective cholecystokinin receptor antagonist. Arch Surg 1990; 125: 1546-1549. GRONROOS JM, KAlLA T, AHO HJ, NEVALAINEN TJ: Decrease in the number of muscarinic receptors in rat pancreas after chronic alcohol intake. Pharmacol Toxicol 1989;64: 356-359. GRONROOS JM, LAINE J, KAlLA T, NEVALAINEN TJ: Chronic alcohol intake and carbachol-induced acute pancreatitis in the rat. Exp Toxic Patholl994; 46: 163-167. HUUPPONEN R, SAXEN U, KouLU M: The efficacyof glibenclamidein rats with type II diabetes induced by neonatal streptozotocin. Diabetes Res 1992;21: 59-63. KONRAD RJ, JOLLY YC, MAJOR C, WOLF BA: Carbachol stimulation of phospholipase A2 and insulin secretion in pancreatic islets. Biochem J 1992; 287: 283-290. OTSUKI M, NAKAMURA T, Y. OKABAYASHI Y, et al.: Effect of a new cholinergic agonist,aclatoniumnapadisilate, on exocrine and endocrine rat pancreas. Dig Dis Sci 1989; 34: 1249-1256. TURK J, GROSS RW, RAMANADHAM S: Amplification of insulin secretion by lipidmessengers. Diabetes 1993; 42:367-374. VERCHERE CB, KWOK YN, BROWN JC: Modulation of acethylcholine-stimulated insulin release by glucose and gastric inhibitory polypeptide. Pharmacoll99l; 42: 273-282.
Exp Toxic Patho! 48 (1996) !
79