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Indian Journal of Experimental Biology Vol. 39, May 2001, pp. 416-424 Pancreatic polypeptide cells of rat pancreas after chronic ethanol feeding Yesna Kokol.2, Vera Todorovicl.2··, Neda Drndarevic 1, Radmila Glisic3 , Miroslava Nedeljkovic 2 & Anna Nikolic 4 'Institute for Medical Researc h, Belgrade, Yugoslavia Institute of Zoology, Faculty of Biology, Belgrade, Yugoslavia 3 lnstitute of Zoology, Faculty of Science, Kragujevac, Yugoslavia 4 lnstitut for the Application of Nuclear Energy, Zemun, Yugoslavia 2 Received 15 October 1999; revised 23 January 200 I Male Wistar rats, (2 months old) were randomly divided into two groups accordi ng to the diet offered (C-control and Ecthanol treated rats). Final body weight was significantly increased but pancreatic weight as a percentage of body weight 2 was decreased in ethanol treated rats. Volume density, number of pancreatic poly peptide (PP)-cells per islet and per ~lm of islet were significant ly increased. PP-cells were abundant and occ upied the whole periphery of islets in the splenic part of the pancreas. Those cells showed strong immuno posit ivity. At the ultrastructural level PP granules had predominantly less electron density. The mean diameter of PP g ranules was signifi ca ntly increased and the number of granules of larger diameter was greater in theE g roup of rats, than in the controls. Pancreatic polypeptide (PP) is a 36 amino acid hormone belonging to a family of structurally related peptides including peptide YY and neuropeptide yu_ PP is synthesized in and released from endocrine PP cells situated within the pancreatic islets and scattered throughout the exocrine tissue 3 . The relative proportions of different pancreatic islet cells show regional and species differences. ]n both the human and rat pancreas, two distinct types of islets have been described, differing from each other in their cellular content: a PP-rich , glucagon-poor islet (PP islet) characteristic of the ventral lobe (derived from the ventral primordium) and glucagon-rich, PPpoor islet (glucagon islet) characteristic of the dorsal primordium4. Under physiological circumstances, PP is mainly secreted in response to ingestion of a meal. The physiological function of the peptide is poorly understood, although it has been demonstrated that under experimental conditions, the peptide inhibits the secretion of insulin in rats, dogs and mice, and inhibits exocrine pancreatic secretion in man and in dogs 5. *Correspondent author. Institute for Medi cal Research DR Suboti a 4, PO BOX 102 11129 Belgrade, Yugoslavia Tel: 381 II 685 788. 381 II 684 484 Fax: 381 II 643 691. e-mail: [email protected] The secretion of PP is predominantly regulated by the parasympathetic nervous system 6·7 • The basal level of serum PP has become a field of interest mainly due to the role of PP as a tumour marker8. 10 , and as a marker of pancreatic endocrine function. In comparison with general mucosal alteration of the gastrointestinal tract, relatively little is known about the effect of alcohol on the diffuse neuroendocrine system (ONES) and its neurohormonal response11.14. Alcohol-associated effects are either due to direct local action or mediated by numerous other substances 15.20 . Although controversy regarding the definition of different histological types of alcoholic chronic pancreatitis in humans and animal models prevails in the literature 21 , the association between ethanol and chronic pancreatitis is well established. Functional studies have shown that exocrine deficiency has a significant effect on islet cell function, and there are exocrine pancreas-insulin, glucagon, pancreatic polypeptide and somatostatin interactions, as well as enteropancreatic interaction in chronic pancreatitis22'23. To the best of our knowledge, complex quantitative analysis of the light microscopic and fine structure of islet endocrine cells has not been carried out in chron ic alcoholism, especially on the PP-cells. The aim of this study is to examine PP-cells at the light and ultrastuctural levels in rats after long term (4 months) alcohol ingestion. KOKO eta/. : PANCREATIC POLYPEPTIDE CELLS AFTER CHRONIC ETHANOL FEEDING Materials and Methods Animals and f eeding protocol - Male Wi star rats (2 month s old) , weighing approximately 240g were randomly allocated to two groups (C-control; E-ethanol treated animals). The control group of 15 rats was fed a commercial diet. The E group of 14 rats was given free access to the diet and a solution of 25% sucrose-32% ethanol as recommended by Hartroft 24 . The quantity of food offered to the control animal s was adjusted to the energy intake of anim als receiving alcohol (i.e. control rats were pair fed). The amount of tap water given to the eth anol-treated rats was 25 ml , and the mean co nsumption per rat per day of th e alcohol solution was 12 ml per ani mal. Ethanol treated rats therefore cons umed 23% of th e total calories as alcohol (mean 8.42 g/kg/day). Blood ethanol determination - Samples for blood eth anol determin ation were obtained from the tail vein, and blood ethanol concentration was determined using the Sigma diagnostic alcohol procedure (No. 322-UY, Si gma Chemical Co., St. Loui s, Mo) . Mean blood ethanol concentration was calculated from multiple blood determinations (at 1000 hrs on day 1, 15, 30, 45 , 60, 75, 90, 105 and 120) throughout the exposure period of 120 days. Blood glucose and plasma insulin determination Fasting blood glucose level was measured by the glucose oxidase method 25 . Plasma insulin was measured by radioimmunoassay usi ng commerci al kits in accordance with the instructions (INEPDiagnostics, Ze mun ) and rat insulin standard (Novo Industry, Bagsvaerd, Denmark) . Preparation of histological samples - After 4 months, rats were starved for 24 hr and given water ad libitum before being killed in the morning (0700 hrs) by a blow on the neck. The pancreas was removed, weighed in air, and the splenic part of the pancreas was cut and used for light and electron microscopic investigation. Samples were immersed for 24 hr in Bouin fixative and embedded in paraffin according to the standard procedure. Immunohistochemistry was performed in 5 J..lm serial section s using polyclonal antibodies agai nst somatostatin, insulin, glucagon and pancreatic polypeptide. Sections were immunostained employing the streptavidinbiotin or PAP technique. Convention al morphometry and standard stereological equations were used to calculate the volu me density and frequency of pancreatic endocrine cells26 . 417 Electron microscopy - Several pieces of the splenic part of the pancreas of each animal were fixed in 2.5% glutaraldehyde in 0.1 M caccodylate buffer and postfixed in 2% osmium tetroxide in the same buffer. After dehydration the specimens were cut on an LKB ultratome, double stained with uranyl acetate and lead citrate and examjned under Philips CM 12 electron microscope. Statistics- Data were subjected to Student's t-test and the results expressed as mean± SE. Results There was no significant difference between the eth anol-treated and control rats with regard to dail y energy intake and energy from protein and fat (Fig. 1). The average ethanol intake of group E was 3.09 g/day which was 23 % of the daily energy intake. Mean blood ethanol concentration was 125 ± 13 mg/ml and ranged from 107 to 138 mg/100 mi. Animals in the examined groups had similar weight gains (data not shown) and body mass increased du ring the experiment continually . Total pancreatic 380 KJ 392 KJ RO ~ ~ c .~ "0 ~ (jt) Ol c -m c ....0 ·;:: 5 40 c ~ "' :J u ~ "' n.. 2C ethanol control []=rJ protein - fat c:::::::::J carl>ohyd. c:::::::::J ethanol Fig. !- Daily energy intake and percentage of particular nutri tional ingredients in the rats of groups C and E. INDIAN J EXP BIOL, MAY 2001 418 weight and volume were similar in both groups, but relative pancreatic weight in rats of group E was decreased (Table I). The results for fasting blood glucose and insulin level were similar in the two groups of animals. There was significant a difference between the ethanol-treated rats and controls with regard to fin al body weight. Volume density, number of PP-cells per islet and per Jlm 2 of islet were significantly increased (Table 2). Mean profile area of the PP-cell was similar in both groups of rats. The number of PP-cells in the exocrine part of the pancreas was reduced, but statistically significant differences were not found. Glucagon-producing A-cells, somatostatinproducing 0-cells and pancreatic polypeptideproducing PP-cells were increased in number, while insulin-producing B-cells were decreased (Table 3). Results obtained for individual endocrine cells per islet showed changes in the percentage of those cells in the E group of rats (Fig. 2). The percentage of Aand PP-cells was increased and that of B-decreased in ethanol-treated rats. The percentage of 0-cells was unchanged. In comparison with the control, the increase in number of PP-cells amounted to about 42% 2 per islet and 83 % per Jlm of islet. It was observed that PP-cells were situated peripherally on the islets singly or in small clusters in the C group of rats while in some islets of Langerhans there were no PP cells. In the exocrine part of the pancreas, pancreatic polypeptide-producing-PP-cells were found both in the acini and in the small ducts. In ethanol-treated rats PP-cells were abundant and occupied the whole periphery of the islets (Fig. 3c). There were no islets without PP-cells unlike in the control (Fig. 3b). In the exocrine tissue PP-cells were Table 1- found in the acini and in the small ducts (Figs 4a,b). A great number of PP-cells had very strong immunoreactivity suggesting accumulation of pancreatic polypeptide granules in the cytoplasm. Electron microscopic examination showed that PPcells in alcohol-treated rats con tained granules in the whole cytoplasmic area. PP granules had less dense cores and closely fitting limiting membranes Table 2-Volume density, number or PP cell s and mean profile areas of PP cells [Values are mean± SE] GroupE Group C Volume density (m m2) 0.!3±0.015 0.24±0.030** Npplprolile area of islet 11.4 ± 1.64 16.7 ± 1.76 Nppl!ln/ of islet 0.0012 ± 0.0001 0.0022 ± 0.0002** NPP in the exocrine pan of pancreas 25.6 ± 4.91 19.8 ± 2.92"' Npplmm 2 of ex.ocrine pancreas 1.36 ± 0.24 1. 155 ± 0. 18"' Mean profile area of PP cell 88.96±3.67 P values:** <0.00 1; "' not significant 87. ! 3 ±4.95'" Table 3-Number of part' cuiar re llslislet Cell type c E a 21.2± 1.95 27. 1 ± 3.75"' ~ D 30.8 ± 1.48 25.5 ± 2.73"' 3.7 ± 0.38 4.2 ± 0.55"' 11.0 ± 1.52 16.7 ± 1.75* pp P values: * < 0.05; "'not signifi cant Body and pancreatic weight, total pancreatic vo lume and fasting blood glucose and insulin level in the rats [Values are mean± SE fro m 15 animals] Group C GroupE Initi al body weight (g) 232±1.27 240±2.37'" Final body weight (g) 359±5.61 426±!8.00** Pancreati c weight (g) 1.30±0.039 1.33±0.073"' Pancreatic weight (% of body weight) Total pancreatic volume (cm 3 ) 0.36±0.012 0.32±0.014* 1.24±0.037 1.26±0.070"' Glucose (mmol/L) 5.28±0.26 5.36±0.13"' Insulin (mlU/L) 22.0±1.34 2!.5±3.06"' P values:** <0.001; * <0.02; "' not significant KOKO eta/.: PANCREATIC POLYPEPTIDE CELLS AFTER CHRONIC ETHANOL FEEDING 419 (Figs 5a,b ). The mean diameter of PP granules was significantly increased (209 ± 3.8 in the control and 224 ± 4.0 in theE group of rats). Although, the largest number of PP granules was found in the same diameter group in both groups of rats (222 nm), in the ethanol treated group the number of granules of larger diameter was greater (Fig. 6). Discussion In a series of experiments, started in 1989, we have been investigating the diffuse neuroendocrine system (ONES) in the upper part of the digestive tract and pancreas after long-term (4 or 6 months) ethanol consumption with or without adequate protein in the diet. Our study mainly consisted of morphofunctional and stereological investigations at the light and electron microscopic level because there are few data about this problem. In the present study, as we11 as in previous investigations we applied a nutritional schedule that made it possible to obtain similar mean daily energy intakes in both groups of rats. Rats given a chronic dose of ethanol (about 23 % of total calories) consumed an adequate amount of other nutrients and had a normal growth rate when compared to pair fed controls. In this respect, the ethanol diet was nutritionally adequate, and possible effects of alcohol were not confounded with the effects of malnutrition 12.24.27.28 . There are no data in the available literature about the detailed morphometric parameters of the PP-ce11s after long-term of ethanol ingestion in rats and our results are the first in this area of investigation. Owiang et al. 23 showed that the number of PP-ce11s so ~ ]:! .'.!! .,... a. "0 ., U) U) ~ .,a. )( !!1 r;; .,uc: u 0 "0 c: UJ A cells B cells D cells PP cells Fig. 2-Percentage of each endocrine cell expresed per islet Fig. 3-(a) Splenic part of pancreas in control rat. Small number of PP cells situated peripherally and islet without PP cells; PAP; x 20; (b) Splenic part of pancreas in ethanol treated rat. Abundant PP cells occupied all periphery of islet; PAP; X 40 420 INDIAN J EXP BIOL, MAY 200I 23 appear to increase in chronic alcoholic pancreatitis . Islets were composed largely of PP-cells, and a large number of isolated PP-cells were frequently found in 29 close contact with duct epithelium . This observation suggests that the increase in PP-cells may represent a regenerative phenomenon after pancreatic injury. There was morphological evidence for an alcoholinduced selective intrapancreatic nerve degeneration in mice after 4 months of alcohol consumption. This affected mainly the nerve fibers that are inhibitory to the exocrine pancreas, and may represent the morphological background of the hypersecretory state of the pancreas in chronic alcoholism. A sli ght decrease was found in the intensity of VIP and SP immunoreactivity and the PP fibers almost di sappeared. Majority of periacinar nerve terminals showed degenerative changes whereas the perivascular nerve fibers were relatively well preserved 30 . Fig. 4-(a) PP cells in the acini. PAP; x 40; (b) PP cells in the small duct: PAP; x 40. The present results showed a significant increase in number of PP-cells in the islets of Langerhans after 4 months consumption of ethanol. It is not clear what is the function al significance of this increase. Other results obtained in the present experiment showed that the number of A-and D-cells was slightly increased while that of B-cells decreased. In addition, some morphological abnormality of A-and B-cells was recorded. T he profile area of A-cells was small er, as well as the profil e area and volume densi ty of th eir 31 nuclei . The B-cells appear more affected and sensitive than the other endocrine cells in the islets 32 . Both B-and A-cells showed degenerative processes suggestive of apoptotic death. The D-and PP-cells in the islets of Langerh ans did not express simil ar morphological changes. Previous results for other endocrine cells belonging to DNES also showed changes in their number12"14"31J2.33. Thus all endocrine cells in th e fundic and pyloric part of the sto mach were significantly decreased in number and some cytological abnormality was also observed. In the rat pancreas glucagon and pancreatic polypeptide are present in di sti nct cell types, but they also coexist in one population of islet cells termed glucagon/PP-cells. The glucagon/PP-cells were found in significant numbers in all pancreatic regions, being more numerous than the other two types of PP-and Acells34. Many of the PP granules in ethanol treated rats were electron pale. It seems th at these PP-cells pro35 duced predominantly pancreatic polypeptide . In this context present results showed that there was proliferation of these cells, although no mitoses was observed in the PP-cells. A possible explanation may be that the population of cells with coex istence of both peptides changed their genetic programme and began to produce predominantly pancreatic polypeptide. The increased diameter of PP granules in the E group of rats probably results fro m accumu lation of pancreatic polypeptide. Functional studies using various tests for insulin secretion have been performed both in patients and animals in chronic pancreatitis. Fasting insulin levels in chronic pancreatitis were found to be normal or 22 moderately raised 36·37 or diminished and the re38 36 38 sponse to oral glucose was variable · . Folch et at. reported that blood glucose concen trations were higher in patients with pancreatitis when compared to controls 38 . On the other hand, serum immunoreactive KOKO eta/.: PANCREATIC POLYPEPTIDE CELLS AFfER CHRONIC ETHANOL FEEDING l: Fig. 5-(a) PP granules in control rat; urany l acetate; lead citrate; bar I Jlm; (b) PP granules with less dense cores in alcoholized rat; uranyl acetate; lead citrate; bar= I Jlm. 421 INDIAN 1 EXP BIOL, MAY 2001 422 ___.__co ntrol · ··•··· ethanol (/) ~ 30 :::J 25 .... 0'1 ..... 20 s::: C'll 0 Cll 0'1 -... C'll s::: 15 10 Cll (J Cll c.. 5 0 . 130 148 166 185 203 222 241 259 278 296 315 PP granules diameter (nm) Fig. 6--Size- frequency distribution of the profile diameter of granules inC and E groups of rats insulin (IRI) concentrations were diminished in chronic human alcoholics 39 , while ethanol had no effect on IRI in ethanol-treated rats 40.4 1• Infusion of ethanol at increasing concentrations caused progres42 43 sive inhibition of insulin and glucagon secretion · . 44 However, Jauhonen and Hassinen observed that ethanol itself produced no marked changes in blood glucose, IRI and glucagon after intravenous infusion. The present results showed that there was no hypoglycaemia which is a strong stimulus for production of pancreatic polypeptide. The plasma level of PP increased significantly after intravenous ethanol administration 45 . Ingestion of alcohol by healthy volunteers did not stimulate the release of cholecystokinin (CCK), which is the chief hormonal stimulant of pancreatic enzyme secretion, nor did it significantly alter fasting levels of pancreatic polypeptide46. These data suggest that, in patients who do not have chronic pancreatitis, alcohol does not induce acute pancreatitis, either by stimulating CCK release or by stimulating enzyme secretion directly. On the contrary, in chronic alcoholic pancreatitis, fasting and postprandial levels of pancreatic polypeptide were significantly increased and the link between CCK and pancreatic polypeptide was disrupted 47 . In addition, Hajnal et al. 48 tested the effect of a 12% (v/v) alcoho l solution added to a meal on basal and postprandial levels of PP in both nonalcoholic volunteers and chronic alcoholics 48 . Basal plasma PP levels were similar in both groups, but the postprandial increments in PP levels observed in nonalcoholics were not observed in alcoholics. However, basal and postprandial levels of gastrin and CCK were similar in alcoholics and nonalcoholics. They concluded that postprandial hypersecretion of pancreatic enzymes in alcoholics is not related to increased plasma levels of CCK or gastrin but it is possible that the impaired release of PP may participate in the mechanism for increased pancreatic enzyme secretion in chronic alcoholics48. Hyperplasia of PP-cells in the PP-rich region of the pancreas (ventral embryonic origin) was observed in patients previously treated surgicall y for functioning duodenal or pancreatic gastrinoma not associated with multiple endocrine neoplasia syndrome49 . PP hyperplasia was observed in the pancreas of j uvenile diabetes50. Although hyperplasia of PP-cells took place predominantly in altered exocrine pancreases, in the present study all rats (with or without histological disturbances of the exocrine pancreas) showed hyperplasia of PP-cells only in the islets of Langerhans. Proliferation of PP-cells was also observed in ethanol treated rats which were fed with a low protein di et (data not shown). It may be concluded that ethanol had a direct effect on this proliferation , al though the exact mechanisms of this phenomenon can not be explained at present. Acknowledgement This work was supported by grant 13M 13 from the Serbian Ministry of Science and Technology, Yugoslavia. 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