Originals Basic Increase in Glucose-Stimulated Insulin Release and Insulin Biosynthesis in Isolated Pancreatic Islets from D-Galactosamine-Treated Rats
Summary Insulin secretion in response to glucose, glucose-stimulated insulin biosynthesis and insulin content was studied in pancreatic islets freshly isolated from male Wistar rats (150—200 g) with galactosamine-induced hepatitis. Animals were sacrificed by decapitation 3, 6,12 and 24 hours after a single intraperitoneal injection of 500 mg/kg of galactosamine. Isolated islets prepared by collagenase method were perifused in Swim's medium with 20 mM glucose at 37 °C up to 30 minutes. Samples were taken at 2—10 min intervals for insulin assay. Insulin biosynthesis was assessed by the incorporation of [ H]-leucine into immunoprecipitable products (insulin and proinsulin) in pancreatic islets after 120 min incubation with 20 mM glucose. Glucose-stimulated insulin secretion was significantly increased at 6, 12 and 24 hours following the administration of galactosamine compared to control. The rate of insulin biosynthesis was stimulated to 170, 138 and 185% of control level 3, 6 and 12 hours after galactosamine-treatment, respectively. Significant increase in insulin content of islets was found 24 hours after galactosamine treatment, following the increased insulin biosynthesis.
sitivity in target tissue (Bottermann, Zilker, Ermler, Paterek and Stransky 1978; Chupin, Charbonnel, Bodic, Grolleau, Chupin and Guillon 1978; Kremer, Atzpodien, Jacobi, Friederichs and Friederichs 1976; Record, Alberti, Williamson and Wright 1973). In D-galactosamine-induced experimental hepatitis a reduced number of insulin receptor sites in the liver membrane was shown preceding an increase in plasma insulin level and therefore a decreased insulin degradation in the liver caused by a receptor defect is thought to be the primary event to hyperinsulinemia (Bachmann, Haslbeck, Bottger, Hepp and Mehnert 1979). However, studies on pancreatic B cell function have not been well established in experimental hepatitis. The aim of the present studies was to determine whether or not pancreatic B cell function is activated in acute liver injury. Insulin secretion in response to glucose, glucosestimulated insulin biosynthesis and insulin content were studied in pancreatic islets freshly isolated from D-galactosamine-treated rats. Materials and Methods Animals
The present results indicate that pancreatic B cell function is activated in early stage of acute liver injury. Key words Galactosamine Hepatitis — Isolated Pancreatic Islets - Glucose-Stimulated Insulin Release - Glucose-Stimulated Insulin Biosynthesis
Introduction
Male Wistar rats (150—200 g) were purchased from Japan Clea Co. Ltd., and fed on laboratory chow ad libitum for one week. After an overnight fast, animals were sacrificed by decapitation at 3, 6, 12 and 24 hours following a single intraperitoneal injection of D-galactosamine-HCl (500 mg/kg b.w.). Blood samples were obtained from unanesthetized rats by vena cava inferior puncture. Plasma glucose was determined by the glucose oxidase method. The determination of plasma glutamic pyruvic aminotransferase (GPT) was according to Reitman and Frankel (1957). Commercial Kit (Dinabot Co. Ltd.) was used for insulin (IRI) assay using rat insulin as a standard. Oral glucose tolerance test
Based on previous studies in man an increased plasma insulin or C-peptide level in addition to impaired glucose tolerance after glucose loading has been demonstrated in acute viral hepatitis, suggesting a reactive increase of insulin secretion from pancreatic B cell due to a decreased insulin sen-
Animals were given 2 g/kg body weight of 20% (W/V) glucose solution through a stomach tube after an overnight fast. Heparinized blood was drawn from the tail vein at 0, 30 and 60 minutes after glucose loading for assay of plasma glucose and insulin. During oral glucose tolerance test the rats were kept in a restraining cage in unanesthetized state.
Horm. meta. Res. (1990) 319- 322 © GeorgThieme Verlag Stuttgart-New York
Received: 6 March 1989
Accepted: 20 Oct. 1989 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
H. Senmaru, H. Park, K. Ohohashi, K. Yoshitoku, T. Nakabayashi, H. Tomimasu and K. Kashima Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan
Horm. meta. Res. 22 (1990) Table 1
H. Senmaru, H. Park, K. Ohohashi, K. Yoshitoku, T. Nakabayashi et al.
Fasting plasma glucose and insulin levels after galactosamine treatment.
FPG (mg/dl) IRI (iw/ml)
Control (6)
3 hours (4)
6 hours (4)
93±12 17.5 ±1.8
107 ± 8 19.9 ±1.3
105 ± 3 21.6 ±3.5
12 hours (4) 93±11 21.6 ±3.3
24 hours (4) 108 ± 8 22.8 ±3.0
Values are expressed as mean±SEM, with the number of experiments in parentheses. FPG: Fasting Plasma Glucose.
Fig. 2 Glucose-stimulated insulin release from perifused rat pancreatic islets in columns of Bio-Gel P2 polyacrylamide beads. O — O : control ( n = 8 ) , © — • : 24 hours after D-galactosamine treatment (n = 6). Each point represents the mean ± SEM. Significantly different from control at each point. *P
Summary Insulin secretion in response to glucose, glucose-stimulated insulin biosynthesis and insulin content was studied in pancreatic islets freshly isolated from male Wistar rats (150—200 g) with galactosamine-induced hepatitis. Animals were sacrificed by decapitation 3, 6,12 and 24 hours after a single intraperitoneal injection of 500 mg/kg of galactosamine. Isolated islets prepared by collagenase method were perifused in Swim's medium with 20 mM glucose at 37 °C up to 30 minutes. Samples were taken at 2—10 min intervals for insulin assay. Insulin biosynthesis was assessed by the incorporation of [ H]-leucine into immunoprecipitable products (insulin and proinsulin) in pancreatic islets after 120 min incubation with 20 mM glucose. Glucose-stimulated insulin secretion was significantly increased at 6, 12 and 24 hours following the administration of galactosamine compared to control. The rate of insulin biosynthesis was stimulated to 170, 138 and 185% of control level 3, 6 and 12 hours after galactosamine-treatment, respectively. Significant increase in insulin content of islets was found 24 hours after galactosamine treatment, following the increased insulin biosynthesis.
sitivity in target tissue (Bottermann, Zilker, Ermler, Paterek and Stransky 1978; Chupin, Charbonnel, Bodic, Grolleau, Chupin and Guillon 1978; Kremer, Atzpodien, Jacobi, Friederichs and Friederichs 1976; Record, Alberti, Williamson and Wright 1973). In D-galactosamine-induced experimental hepatitis a reduced number of insulin receptor sites in the liver membrane was shown preceding an increase in plasma insulin level and therefore a decreased insulin degradation in the liver caused by a receptor defect is thought to be the primary event to hyperinsulinemia (Bachmann, Haslbeck, Bottger, Hepp and Mehnert 1979). However, studies on pancreatic B cell function have not been well established in experimental hepatitis. The aim of the present studies was to determine whether or not pancreatic B cell function is activated in acute liver injury. Insulin secretion in response to glucose, glucosestimulated insulin biosynthesis and insulin content were studied in pancreatic islets freshly isolated from D-galactosamine-treated rats. Materials and Methods Animals
The present results indicate that pancreatic B cell function is activated in early stage of acute liver injury. Key words Galactosamine Hepatitis — Isolated Pancreatic Islets - Glucose-Stimulated Insulin Release - Glucose-Stimulated Insulin Biosynthesis
Introduction
Male Wistar rats (150—200 g) were purchased from Japan Clea Co. Ltd., and fed on laboratory chow ad libitum for one week. After an overnight fast, animals were sacrificed by decapitation at 3, 6, 12 and 24 hours following a single intraperitoneal injection of D-galactosamine-HCl (500 mg/kg b.w.). Blood samples were obtained from unanesthetized rats by vena cava inferior puncture. Plasma glucose was determined by the glucose oxidase method. The determination of plasma glutamic pyruvic aminotransferase (GPT) was according to Reitman and Frankel (1957). Commercial Kit (Dinabot Co. Ltd.) was used for insulin (IRI) assay using rat insulin as a standard. Oral glucose tolerance test
Based on previous studies in man an increased plasma insulin or C-peptide level in addition to impaired glucose tolerance after glucose loading has been demonstrated in acute viral hepatitis, suggesting a reactive increase of insulin secretion from pancreatic B cell due to a decreased insulin sen-
Animals were given 2 g/kg body weight of 20% (W/V) glucose solution through a stomach tube after an overnight fast. Heparinized blood was drawn from the tail vein at 0, 30 and 60 minutes after glucose loading for assay of plasma glucose and insulin. During oral glucose tolerance test the rats were kept in a restraining cage in unanesthetized state.
Horm. meta. Res. (1990) 319- 322 © GeorgThieme Verlag Stuttgart-New York
Received: 6 March 1989
Accepted: 20 Oct. 1989 after revision
Downloaded by: University of Pennsylvania Libraries. Copyrighted material.
H. Senmaru, H. Park, K. Ohohashi, K. Yoshitoku, T. Nakabayashi, H. Tomimasu and K. Kashima Third Department of Internal Medicine, Kyoto Prefectural University of Medicine, Kyoto, Japan
Horm. meta. Res. 22 (1990) Table 1
H. Senmaru, H. Park, K. Ohohashi, K. Yoshitoku, T. Nakabayashi et al.
Fasting plasma glucose and insulin levels after galactosamine treatment.
FPG (mg/dl) IRI (iw/ml)
Control (6)
3 hours (4)
6 hours (4)
93±12 17.5 ±1.8
107 ± 8 19.9 ±1.3
105 ± 3 21.6 ±3.5
12 hours (4) 93±11 21.6 ±3.3
24 hours (4) 108 ± 8 22.8 ±3.0
Values are expressed as mean±SEM, with the number of experiments in parentheses. FPG: Fasting Plasma Glucose.
Fig. 2 Glucose-stimulated insulin release from perifused rat pancreatic islets in columns of Bio-Gel P2 polyacrylamide beads. O — O : control ( n = 8 ) , © — • : 24 hours after D-galactosamine treatment (n = 6). Each point represents the mean ± SEM. Significantly different from control at each point. *P
