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Pancreatic exocrine dysfunction in a model of spontaneous type I diabetes
April 1998 agonists. Total RNA was isolated by a modified single-step method. Mob-1 mRNA levels were measured using Northern-blotting. NF~:B activity was estimated by measurement of the loss of the inhibitory subunit I~:B-ct using Western-blotting. Results: In the current study care was taken to handle the acini gently during isolation and the cells were incubated without shaking to avoid mechanical stress. Under these conditions there was little or no mob-1 expression in freshly isolated acini and mob-1 gene expression was increased by CCK 8 in a dose- and time-dependent fashion. Mob-1 gene expression was increased by hyperstimulatory concentrations ( > 1 nM) but not by physiologic concentrations of CCK. CCK induced mob-1 expression was significant within lh and maximal by 2 h. Neither bombesin nor carbachol significantly increased mob-1. Acinar cell IKB-~t levels were also decreased by CCK 8 in a dose- and time-dependent fashion. A significant decrease in IKB-ct was noted with 1 nM and maximal effects were observed with 100 nM CCK 8. Decreases in I~:B-ct were noted within 30 minutes and maximal after 2 hours. Thus, the concentration- and time-dependence for mob-1 gene expression and I~:B-~t degradation were similar. Conclusions: Rat pancreatic acini isolated by a modified method are a useful model for the study of the mechanisms of mob-1 gene expression. In this model CCK induced activation of chemokine gene expression and NFtzB activation showed parallel concentration- and time-dependence. This suggests that the NF~B signaling pathway may be important for regulating pancreatic chemokine expression and play a role in acute pancreatitis. G1899 ESWL TREATMENT OF PANCREATIC DUCT STONES IN SYMPTOMATIC CHRONIC PANCREATITIS. S Hansen, B Brand, VC Nam, KF Binmoeller, U Seitz, S Bohnacker, F Thonke, N Soehendra, Department of Endoscopic Surgery, University of Hamburg-Eppendorf, 20246 Hamburg, Germany Backeround: Occlusion of the main pancreatic duct by stones and strictures may cause increased outflow pressure and is believed to be one pathogenic factor underlying the pain associated with chronic pancreatitis (CP). In these patients (pts.) ESWL and pancreatic stenting have been introduced as new tools for effective symptom relieve. Methods: We report our results of ESWL used alone or with pancreatic stenting. Between 1/1990 and 6/1996, 169 pts. with symptomatic CP and duct stones with/without strictures were referred to our department and received ERP pre- and post ESWL. ESWL was performed with a modified lithostar prototype with electromagnetic shockwave generation (Fa. Siemens). Targeting was performed by ultrasound. Results; Etiology of CP was alcoholic in 54.3%, idiopathic in 44.2%, others 1.5%. Duration of symptoms pre-ESWL was 156 months (median, range: 0-300). Localisation of stones was restricted to the head of the pancreas in 120 of the cases. 73 pts. had a solitary stone, 96 pts. had multiple stones. The total amount of shockwaves was 6600 (median, range: 2000-42200). No analgosedation was necessary. All but 5 pts. recieved EPT prior or during ESWL-treatment. Fragmentation rate of the stones was 100% (169/169) according to the ultrasound-controls during ESWL treatment. After a median of 7 sessions (range: 3-32), stone free rate was 73.4% (124/169), remaining fragments were seen in 21.8% (37/169), visualization of the pancreatic duct was not achieved in 4.7% (8/169). In 50% (8/16) of the cases with pancreatic stenting prior to ESWL (group A) the stents were not tolerated during ESWL. Following ESWL, pancreatic stenting was performed in 33/169 pts. (group B) with a dominant proximal stricture of the main pancreatic duct. In all but 2 (6%) of these pts., stenting was now tolerated well (group A vs. group B, Chi square test: p < 0,001). All but one patient with a follow up > 12 months (n=91) experienced pain relief. If pts. with follow up < 12 months are included, symptom relief was achieved in 121/140 (86.4%) pts.. Pancreatic surgery was performed in 19/142 (!3.4%) cases due to persistent or recurrent pain. Relapsing symptomatic stones were seen in 5 pts. after 22 months (range: 5-36). Symptom relief and stone clearance were not correlated with stone number or localisation, presence of parenchymal calcifications, strictures or stenting (Chi square test). Pain aggravation caused termination of ESWL in 3 pts. and temporary interruption of ESWL in 5 pts.. ERP related complications occured in 2 pts., one bleeding after EPT, one acute pancreatitis after ERP. Conclusion: ESWL offers a safe and effective treatment of symptomatic pancreatic duct stones. Surgery, influencing life quality and including high operative risks, can be avoided in the majority of cases. • G1900
LIPID STIMULATED SECRETION OF CCK IS MEDIATED BY CCKRELEASING PEPTIDE (DIAZEPAM BINDING INHIBITOR): INVOLVEMENT OF MUCOSAL SEROTONIN AND SUBMUCOSAL SUBSTANCE P AND CHOLINERGIC NEURONS. Y Hao, Y Li, C Owyang. Dept of Int Med, University of MI, Ann Arbor, MI 48109. Lipids in the intestine is a major stimulant of CCK secretion in humans, dogs and rats. Recently we have isolated a trypsin-sensitive CCK-releasing peptide (CCK-RP) from porcine intestinal mucosa. This peptide has an amino acid sequence identical to the porcine diazepam binding inhibitor (DBI) (PNAS 1996; 93:7927) and appears to mediate CCK secretion in the rat. We hypothesized that lipids in the intestine may stimulate the release of CCK via the secretion of CCK-RP (DBI). To test this hypothesis we investigated the effects of intraduodenal administration of oleic acid on luminal DBI secretion, CCK release and pancreatic protein output. After 5 hr of bile-pancreatic juice diversion (when plasma CCK levels had returned to basal levels), intraduodenal perfusion of 10% oleic acid caused a 75 -+ 6% increase in
Pancreatic Disorders A467
protein secretion and an elevation of plasma CCK levels from 0.8 -+ 0.3 to 7.4 -+ 1.6pM. This was accompanied by an increase in luminal DBI secretion from 2.0-+ 1.2 ng/hr to 12-+1.5 ng/hr in the intestinal washings. Immunoneutralization of luminal DBI with rabbit DBI antiserum abolished the stimulatory action of oleic acid on pancreatic protein secretion and plasma CCK levels but had no effect on protein secretion stimulated by maltose, which acts via a CCK-independent pathway. This suggests that DBI mediates oleic acid stimulated CCK secretion. To investigate the neural pathways responsible for the secretion of luminal DBI we showed that the stimulatory actions of oleic acid on DBI secretion and plasma CCK levels were markedly reduced or abolished by atropine or hetamethonium but not vagotomy. Mucosal application of lidocaine but not serosal application of benzalkonium chloride, which ablates the myenteric neurons, also abolished the actions of oleic acid. This suggests that the enteric circuitry controlling DBI secretion is present in the submucous and not the myenteric plexus. To further characterize the local neural reflex in the submucous plexus which mediates DBI secretion, we demonstrated that pretreatment of the rats with the 5HT3 antagonist. ICS-205, 930 and the sub P antagonist CP96,345 (2.5 mg/kg ip) also prevented the secretion of DBI. These observations suggest that oleic acid in the duodenum stimulates serotonin release from the mucosal EC cells, and this activates the sensory substance P neurons in the submucous plexus. Signals are then transmitted to cholinergic interneurons and to epithelial DBI containing cells via cholinergic secretomotor neurons. This enteric neural circuitry which is responsible for the secretion of DBI may play an important role in the release of CCK stimulated by lipids. G1901 PANCREATIC EXOCRINE DYSFUNCTION IN A MODEL OF SPONTANEOUS TYPE I DIABETES. J.A. Hardin J. Fung, L. Donegan, D.G. Gall. Gastrointestinal Research Group, University of Calgary, Calgary, Alberta, Canada. The Bio-Breeding (BB) rat is a well established model of spontaneous Type I diabetes characterized by the autoimmune destruction of pancreatic islets resulting in impaired pancreatic endocrine function. The effect of pancreatic autoimmune inflammation on pacreatic exocrine function is not known. Aim: The aim of this study was to examine pancreatic exocrine function in the spontaneously diabetic Bio-Breeding rat. Methods: Diabetic BB rats (D), prediabetic BB rats (PD), and non-diabetic BB control rats (C) derived from the same colony were studied. Diabetic rats were sacrificed 2-3 days after developing glycosuria and age and weight matched to pre-diabetic and control animals. Pancreatic tissue was obtained for determination of amylase and trypsin activity and tissue fixed for histological assessment of insulinistis on a four point scale. Results: Diabetic animals displayed a significant degree of pancreatic insulinitis (D 1.6 -+ .5 out of 4). Control and pre-diabetic animals had no evidence of any pancreatic inflammatory inflitrate. Diabetic animals had significantly decreased amylase activity in pancreatic homogenates (C 4.5 -+ .6; n=5, vs PD 3.8 -+ .8; n=5, vs D 1.4 -+ .2 U/mg P; n=6) compared to control (p <0.001) and pre-diabetic (p <0.05) animals. Conclusion: The defect in pancreatic endocrine function is associated with impaired pancreatic exocrine function in a genetic model of spontaneous type ! diabetes. • G1902 CYTOKINES AND PROCALCITONIN IN ERCP-INDUCED PANCREATIC DAMAGE. P. H a r d t , O. Kress, T. Fadgyas, M. Oezcueruemez*, D. Kunz*, S. Westphal*, C. Luley*, H.U. Kloer. III. Dept. of Internal Medicine, University of Giessen, Germany; Dept. of Clinical Chemistry, University of Magdeburg, Germany Post-ERCP pancreatitis has been suggested as a model for acute pancreatitis (AP), which allows evaluation of early alterations in the time course of the disease. Procalcitonin (PCT) as a new prognostic parameter for severe bacterial infection has recently been reported to discriminate patients with acute biliary pancreatitis. Several cytokines and anticytokines were also discussed to provide more specific information in the diagnosis of AP. Blood samples of patients with ERCP were prospectively collected to further evaluate the importance of this parameter and its determinants in AP. The incidence of ERCP induced pancreatic damage, defined as a three-fold increase of serum lipase, elevation of CRP from < 10 to > 20 mg/I and abdominal complaints was 12,8% (12/94). Serum levels of PCT, IL-1RA, IL-6, IL-10, TNF¢ and sTNFccRII were measured before, 40 rain, 2 h, 6 h, 24 h, 48 h and 72 h after ERCP in these 12 patients. PCT significantly increased in subjects with post-ERCP pancreatitis after 24h. However, absolute levels did not exceed 0,5 ng/ml in any patient. IL-1RA began to differ from baseline 2h after ERCP, followed by IL-6 (6h) and sTNFc¢ RII (24 h). TNF~ was not detectable in any patient. IL-10 showed high interindividual variations with no obvious peak. Among all evaluated parameters, CRP and lipase included, only peak values of IL-6 and IL-10 showed a significant correlation with the reported pain score (r=0,65/0,78), the number of cannulations (r=-n.s./0,87) and the duration of endoscopy (r=-0,58/0,76). The volume of injected contrast agent showed no significant correlation. We conclude that PCT did not reach clinically relevant levels ( > lng/ml), thus, a systemic bacterial component is not obvious in patients with postERCP pancreatitis. IL-10 and IL-6 seem to be useful to assess the iatrogenic damage induced by ERCP.
LIPID STIMULATED SECRETION OF CCK IS MEDIATED BY CCKRELEASING PEPTIDE (DIAZEPAM BINDING INHIBITOR): INVOLVEMENT OF MUCOSAL SEROTONIN AND SUBMUCOSAL SUBSTANCE P AND CHOLINERGIC NEURONS. Y Hao, Y Li, C Owyang. Dept of Int Med, University of MI, Ann Arbor, MI 48109. Lipids in the intestine is a major stimulant of CCK secretion in humans, dogs and rats. Recently we have isolated a trypsin-sensitive CCK-releasing peptide (CCK-RP) from porcine intestinal mucosa. This peptide has an amino acid sequence identical to the porcine diazepam binding inhibitor (DBI) (PNAS 1996; 93:7927) and appears to mediate CCK secretion in the rat. We hypothesized that lipids in the intestine may stimulate the release of CCK via the secretion of CCK-RP (DBI). To test this hypothesis we investigated the effects of intraduodenal administration of oleic acid on luminal DBI secretion, CCK release and pancreatic protein output. After 5 hr of bile-pancreatic juice diversion (when plasma CCK levels had returned to basal levels), intraduodenal perfusion of 10% oleic acid caused a 75 -+ 6% increase in
Pancreatic Disorders A467
protein secretion and an elevation of plasma CCK levels from 0.8 -+ 0.3 to 7.4 -+ 1.6pM. This was accompanied by an increase in luminal DBI secretion from 2.0-+ 1.2 ng/hr to 12-+1.5 ng/hr in the intestinal washings. Immunoneutralization of luminal DBI with rabbit DBI antiserum abolished the stimulatory action of oleic acid on pancreatic protein secretion and plasma CCK levels but had no effect on protein secretion stimulated by maltose, which acts via a CCK-independent pathway. This suggests that DBI mediates oleic acid stimulated CCK secretion. To investigate the neural pathways responsible for the secretion of luminal DBI we showed that the stimulatory actions of oleic acid on DBI secretion and plasma CCK levels were markedly reduced or abolished by atropine or hetamethonium but not vagotomy. Mucosal application of lidocaine but not serosal application of benzalkonium chloride, which ablates the myenteric neurons, also abolished the actions of oleic acid. This suggests that the enteric circuitry controlling DBI secretion is present in the submucous and not the myenteric plexus. To further characterize the local neural reflex in the submucous plexus which mediates DBI secretion, we demonstrated that pretreatment of the rats with the 5HT3 antagonist. ICS-205, 930 and the sub P antagonist CP96,345 (2.5 mg/kg ip) also prevented the secretion of DBI. These observations suggest that oleic acid in the duodenum stimulates serotonin release from the mucosal EC cells, and this activates the sensory substance P neurons in the submucous plexus. Signals are then transmitted to cholinergic interneurons and to epithelial DBI containing cells via cholinergic secretomotor neurons. This enteric neural circuitry which is responsible for the secretion of DBI may play an important role in the release of CCK stimulated by lipids. G1901 PANCREATIC EXOCRINE DYSFUNCTION IN A MODEL OF SPONTANEOUS TYPE I DIABETES. J.A. Hardin J. Fung, L. Donegan, D.G. Gall. Gastrointestinal Research Group, University of Calgary, Calgary, Alberta, Canada. The Bio-Breeding (BB) rat is a well established model of spontaneous Type I diabetes characterized by the autoimmune destruction of pancreatic islets resulting in impaired pancreatic endocrine function. The effect of pancreatic autoimmune inflammation on pacreatic exocrine function is not known. Aim: The aim of this study was to examine pancreatic exocrine function in the spontaneously diabetic Bio-Breeding rat. Methods: Diabetic BB rats (D), prediabetic BB rats (PD), and non-diabetic BB control rats (C) derived from the same colony were studied. Diabetic rats were sacrificed 2-3 days after developing glycosuria and age and weight matched to pre-diabetic and control animals. Pancreatic tissue was obtained for determination of amylase and trypsin activity and tissue fixed for histological assessment of insulinistis on a four point scale. Results: Diabetic animals displayed a significant degree of pancreatic insulinitis (D 1.6 -+ .5 out of 4). Control and pre-diabetic animals had no evidence of any pancreatic inflammatory inflitrate. Diabetic animals had significantly decreased amylase activity in pancreatic homogenates (C 4.5 -+ .6; n=5, vs PD 3.8 -+ .8; n=5, vs D 1.4 -+ .2 U/mg P; n=6) compared to control (p <0.001) and pre-diabetic (p <0.05) animals. Conclusion: The defect in pancreatic endocrine function is associated with impaired pancreatic exocrine function in a genetic model of spontaneous type ! diabetes. • G1902 CYTOKINES AND PROCALCITONIN IN ERCP-INDUCED PANCREATIC DAMAGE. P. H a r d t , O. Kress, T. Fadgyas, M. Oezcueruemez*, D. Kunz*, S. Westphal*, C. Luley*, H.U. Kloer. III. Dept. of Internal Medicine, University of Giessen, Germany; Dept. of Clinical Chemistry, University of Magdeburg, Germany Post-ERCP pancreatitis has been suggested as a model for acute pancreatitis (AP), which allows evaluation of early alterations in the time course of the disease. Procalcitonin (PCT) as a new prognostic parameter for severe bacterial infection has recently been reported to discriminate patients with acute biliary pancreatitis. Several cytokines and anticytokines were also discussed to provide more specific information in the diagnosis of AP. Blood samples of patients with ERCP were prospectively collected to further evaluate the importance of this parameter and its determinants in AP. The incidence of ERCP induced pancreatic damage, defined as a three-fold increase of serum lipase, elevation of CRP from < 10 to > 20 mg/I and abdominal complaints was 12,8% (12/94). Serum levels of PCT, IL-1RA, IL-6, IL-10, TNF¢ and sTNFccRII were measured before, 40 rain, 2 h, 6 h, 24 h, 48 h and 72 h after ERCP in these 12 patients. PCT significantly increased in subjects with post-ERCP pancreatitis after 24h. However, absolute levels did not exceed 0,5 ng/ml in any patient. IL-1RA began to differ from baseline 2h after ERCP, followed by IL-6 (6h) and sTNFc¢ RII (24 h). TNF~ was not detectable in any patient. IL-10 showed high interindividual variations with no obvious peak. Among all evaluated parameters, CRP and lipase included, only peak values of IL-6 and IL-10 showed a significant correlation with the reported pain score (r=0,65/0,78), the number of cannulations (r=-n.s./0,87) and the duration of endoscopy (r=-0,58/0,76). The volume of injected contrast agent showed no significant correlation. We conclude that PCT did not reach clinically relevant levels ( > lng/ml), thus, a systemic bacterial component is not obvious in patients with postERCP pancreatitis. IL-10 and IL-6 seem to be useful to assess the iatrogenic damage induced by ERCP.