Chronic intragastric alcohol exposure causes hypoxia and oxidative stress in the rat pancreas

The effect of chronic enteral ethanol on pancreatic hypoxia was investigated using the hypoxia marker, pimonidazole. Male Wistar rats were fed an ethanol-containing diet for 3 weeks using an enteral model shown to cause pancreatic damage; pimonidazole (120 mg/kg i.v.) was injected 1h before sacrifice. Pimonidazole and 4-hydroxynonenal (an index of lipid peroxidation) adducts were detected immunochemically. Breathing air with low oxygen content (8% O(2)) for 1h increased pimonidazole adduct accumulation approximately 2-fold in pancreata of nai;ve rats, confirming that this technique will detect increases in hypoxia in pancreata. Pancreata of rats fed ethanol began to show signs of damage after 3 weeks. Ethanol feeding also significantly increased pimonidazole adducts in pancreas approximately 2-fold (1 or 3 weeks of ethanol produced similar values). Concomitant with increasing hypoxia in the pancreas, alcohol also caused a significant increase in 4-hydroxynonenal adducts, indicative of increased oxidative stress. These results indicate that chronic ethanol causes hypoxia at the cellular level in the pancreas in vivo; further, the data support the hypothesis that hypoxia is involved in mechanisms of chronic alcoholic pancreatitis.     

Identification and characterization of receptors for secretagogues on pancreatic acinar cells

Acinar cells from guinea pig pancreas possess six different classes of receptors that mediate the actions of various secretagogues on enzyme secretion. Four classes of receptors stimulate enzyme secretion by causing mobilization of adenylate cyclase and increased cellular cyclic AMP. This paper summarizes the results of studies that have employed radiolabeled secretagogues of high specific activity and have measured directly the interaction of secretagogues with their receptors on pancreatic acinar cells.     

Secretagogue-induced membrane alterations in dispersed acini from rat pancreas

Dispersed acini from rat pancreas were used to examine the effects of various pancreatic secretagogues on the fine structure of the acinar cell plasma membrane. With the C-terminal octapeptide of cholecystokinin, the C-terminal tetrapeptide of cholecystokinin, carbamylcholine, bombesin, A23187, vasoactive intestinal peptide or 8-bromo cyclic adenosine monophosphate, concentrations of the secretagogues that caused maximal stimulation of enzyme secretion did not produce alterations of the acinar cell plasma membrane. Supramaximal concentrations of the C-terminal octapeptide of cholecystokinin, the C-terminal tetrapeptide of cholecystokinin or carbamylcholine induced the formation of cytoplasmic protrusions at the basolateral plasma membrane of the pancreatic acinar cell, whereas supramaximal concentration of bombesin, A23187, vasoactive intestinal peptide or 8-bromo cyclic AMP did not alter the morphology of the acinar cell. Effects of the C-terminal octapeptide of cholecystokinin could be detected as early as after two minutes of incubation and these effects progressed for up to 30 minutes of incubation.     

Glucocorticoids effects on exocrine pancreatic secretion in caerulein-induced acute pancreatitis in the rat.

The present work reports on exocrine pancreatic secretion in control rats, adrenalectomized rats and hydrocortisone-treated (10 mg/Kg/d) rats during 7 days, under normal conditions and after induction of acute pancreatitis with caerulein (20 micrograms/Kg) by 4 subcutaneous injections at hourly intervals. Pancreatic secretion was seen to be affected by the procedure of adrenalectomy, which led to a marked reduction in the secretion of proteins and amylase with respect to control values. This was probably due to the decrease occurring in the zymogen granules in the acinar cells of the exocrine pancreas, a phenomenon which also led to a decrease in pancreatic weight observed in these animals. Treatment with hydrocortisone induced a decrease in the secretion of proteins and amylase, as well as an increase in pancreatic weight. This agrees with the accepted hypothesis that large amounts glucocorticoids stimulate the synthesis and storage of proteins in the exocrine pancreas, reducing the secretory phase. The administration of high doses of caerulein under these conditions led to acute pancreatitis in the three groups of animals. This was paralleled by a dramatic decrease in protein and amylase secretion and by severe interstitial edema of the pancreas and by increases in serum amylase values. In the case of the animals treated previously with hydrocortisone, the latter were tripled with respect to the control animals. The conclusion is offered that since the storage of enzyme proteins is governed by glucocorticoids, which furthermore increase the sensitivity of the acinar cells to stimulation by secretagogues, the administration of these substances during the development of pancreatic lesions such as acute pancreatitis is highly compromising to the organism.     

Effect of leptin on insulin, glugacon and somatostatin secretion in the perfused rat pancreas.

We have investigated the effect of rat leptin as well as the 22-56 fragment of this molecule on pancreatic hormone secretion in the perfused rat pancreas. In pancreases from fed rats, leptin failed to alter the insulin secretion elicited by glucose, arginine or tolbutamide, but inhibited the insulin response to both CCK-8 and carbachol, secretagogues known to act on the B-cell by increasing phospholipid turnover. This insulinostatic effect was also observed with the 22-56 leptin fragment. In pancreases obtained from 24-hour fasted rats, no effect of leptin on carbachol-induced insulin output was found, perhaps as a consequence of depressed B-cell phospholipid metabolism. Leptin did not influence glucagon or somatostatin release. Our results do not support the concept of leptin as a major regulator of B-cell function. Leptin inhibition of carbachol-induced insulin output might reflect a restraining effect of this peptide on the cholinergic stimulation of insulin release.     

Effect of pectin on the function of the rat exocrine pancreas

The author studied the effect of three weeks' administration of a 5% pectin concentration in the standard (Larsen) diet on basal and cholecystokinin octapeptide-(CCK8-) stimulated pancreatic exosecretion in rats. Protein, amylase and trypsinogen output determined in pancreatic juice obtained under basal conditions and after CCK8 stimulation of the exocrine pancreas showed no statistically significant differences between rats fed on the standard and the pectin-enriched Larsen diet. Our observations and studies by other authors show that the direct effect of dietary fibre on the exocrine function of the pancreas is an open question as far as further research is concerned.     

Exocrine function of the pancreas in regularly swimming rats

The exocrine function of the pancreas was investigated in the course of adaptation to regular physical activity. In albino rats exercized by swimming for six weeks it has been observed that - in response to an identical physiological stimulation (liquid food via gastric tube) and under ad libitum feeding the rate of secretion and enzyme activity exceeded control values by 100 to 150%; - in pair-fed rats the values of the exercized animals were 50 to 70% higher, and - vagotomy suspended this effect even when food was offered ad libitum. On the basis of these results adaptation to regular exercise involves the exocrine function of the pancreas. In this functional increase both the increased food intake after training sessions and the direct effect of muscular work have a share. For adaptation an intact innervation is necessary.     

Effect of subcutaneous pancreatic tissue transplants on streptozotocin-induced diabetes in rats. III. Distribution of neuropeptides in normal and diabetic (host) pancreas.

This study examines whether there is a change in the pattern of distribution of cholecystokinin-octapeptide (CCK-8), calcitonin-gene-related peptide (CGRP), neuropeptide-Y (NPY), substance P (SP) and vasoactive intestinal polypeptide (VIP) in the pancreas of streptozotocin (STZ)-diabetic (host) rats after subcutaneous pancreatic transplantation. Varicose CCK-8-immunopositive nerve fibres were observed in the wall of blood vessels of both normal and diabetic host pancreata. The density of CCK-8-immunoreactive varicose nerve fibres appeared to have increased in host rat pancreas. CGRP was demonstrated in many nerve fibres located in the wall of blood vessels of both normal and host pancreas. CGRP, however, seemed to be better expressed in the nerves of host pancreas when compared to normal. The pancreata of both normal and diabetic (host) rats contained numerous NPY-immunopositive varicose nerve fibres located in the wall of blood vessels. SP was demonstrated in neurons located in the interlobular areas of normal tissue and in fine varicose nerve fibres of the interacinar region of the pancreas of STZ-induced diabetic rats with SPTG. In normal pancreatic tissue, VIP-immunopositive nerve fibres were observed in all areas of the pancreas. VIP-positive nerve fibres were still discernible especially in the interacinar regions of the pancreas of host rats. In conclusion, the pattern of distribution and density of NPY, SP and VIP in the pancreas of STZ-induced diabetic rats with SPTG is similar to that observed in normal pancreas, but the expression of CGRP and CCK-8 seemed to have increased as a result of transplantation and or diabetes.     

Behavior of cellular cyclic nucleotides after hydrokinetic or ecbolic stimulation of the canine pancreas

The studies deal with the influence of secretin and various ecbolic secretagogues on tissue levels of cAMP and cGMP in vivo and in the isolated perfused canine pancreas. The mutual behaviour of cellular cAMP and cGMP is observed and compared with the time course of the respective secretory events. Synthetic secretin as well as CCK, acetylcholine or Caerulein likewise elevate tissue cAMP and cGMP simultaneously. There exists no difference in the magnitude of increase and in the time course of changes in tissue cyclic nucleotide levels between hydrokinetic and ecbolic stimulation. The rise in cAMP and cGMP coincides with the onset of the respective secretory events and reaches peak values contemporarily to the excretory maxima. The following decrease in tissue cyclic nucleotides approximatively parallels juice or enzyme secretion in the isolated perfused pancreas but differs widely in vivo. Under this condition cAMP and cGMP rapidly fall to basal levels during undiminished excretory function and show a second rise after cessation of the latter. Secretin and various ecbolic secretagogues do not increase tissue content of cyclic nucleotides in the same dose-dependent manner as can be observed with pancreatic secretion. The behaviour of cAMP and cGMP after addition of secretin and CCK or acetylcholine remains widely unchanged during calcium-free perfusion in spite of an extensive excretory inhibition. The corresponding rise in cellular cAMP and cGMP in the sequence of hydrokinetic as well as of ecbolic stimulation points to an analogous intracellular mediation of various secretagogues in different target cells of the exocrine canine pancreas.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional L-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of L-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (less than 30 s) paired-tracer dilution technique. In the non-diabetic pancreas L-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 microU/ml). Transport of L-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of L-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

Abnormalities of caerulein- and carbamylcholine-stimulated pancreatic enzyme secretion in the obese Zucker rat

The secretory function of the exocrine pancreas has been studied in dispersed pancreatic acini from obese and homozygous lean Zucker rats at 6 and 22 wk. No abnormality was found in acini from young rats. Acini from 22 wk obese and lean rats were equally responsive to secretagogues which stimulate cAMP, i.e. vasoactive intestinal peptide (VIP) and secretin. By contrast, there was a reduction in the maximum responsiveness to caerulein and carbamylcholine in acini from obese rats. These latter secretagogues act through mobilization of intracellular Ca2+. Since obese animals are insulin resistant and amylase release is modulated by insulin, the role of insulin resistance in the secretory defect was then investigated. A group of 22 wk obese rats received treatment with Ciglitazone (a drug which reduces insulin resistance in obese laboratory animals) for 4 wk before the secretion study. Despite the expected reduction in insulin resistance there was no improvement of the secretory defect seen with caerulein and carbamylcholine stimulation. Thus, the secretory abnormality in the exocrine pancreas of adult obese Zucker rats does not appear to be directly associated with insulin resistance. Furthermore, the secretory defect is linked to those secretagogues which induce Ca2+-independent phosphoinositide hydrolysis and Ca2+ mobilization in the target cell.     

EFFECT OF PANCREOZYMIN ON RAT PANCREATIC ENZYME BIOSYNTHESIS

Pancreatic enzyme secretion in rats anesthesized by pentobarbital was stimulated by intravenous perfusion of the hormone pancreozymin, as indicated by a decreased amylase level in the pancreas and by specific, fine structural changes observed in an electron microscope. Rates of protein synthesis were determined by pulse labeling. Amylase, total protein, and valine were purified from pancreas and counted. Pancreozymin promotes an 8 to 10 times increase in the rate of biosynthesis of pancreatic enzymes, as compared to rats similarly anesthesized but without hormone. This stimulation effect is obtained very rapidly (2 hr) and is not inhibited by actinomycin D. Secretin alone has no effect, whereas pentobarbital is inhibitory.     

mRNA expression of pancreatic enzyme precursors and estimation of protein digestibility in first feeding larvae of the Japanese flounder,Paralichthys olivaceus

An understanding of digestibility in marine fish larvae is required to formulate a diet to replace zooplankton. Using flounder, this study was aimed at determining which digestive enzymes are synthesized in the larval pancreas, and how the proteins are cleaved in the digestive canal. Whole mount in situ hybridization indicated that the mRNA of all digestive enzyme precursors examined, including trypsin, chymotrypsin, elastase, carboxypeptidase A and B, and lipase, was expressed in the pancreas of first feeding larvae at 3 days post-fertilization. In the larvae before differentiation of the stomach, protein digestion in the digestive canal mainly depends on pancreatic proteases. So, to evaluate protein digestibility in the larval digestive canal, the digestion of proteins by pancreatic extract was monitored by gel electrophoresis. It was indicated that thyroglobulin, albumin and lactate dehydrogenase were rapidly cleaved to polypeptide fragments, but ferritin and catalase exerted resistance to proteolysis, suggesting that digestibility in the larval digestive canal differs depending on protein species.     

The relationship between beta cell activation and SLC30A8/ZnT8 levels of the endocrine pancreas and maternal zinc deficiency in rats

ObjectivesZinc, which is found in high concentrations in the β-cells of the pancreas, is also a critical component for the endocrine functions of the pancreas. SLC30A8/ZnT8 is the carrier protein responsible for the transport of zinc from the cytoplasm to the insulin granules. The aim of this study was to investigate how dietary zinc status affects pancreatic beta cell activation and ZnT8 levels in infant male rats born to zinc-deficient mothers.MethodsThe study was performed on male pups born to mothers fed a zinc-deficient diet. A total of 40 male rats were divided into 4 equal groups. Group 1: In addition to maternal zinc deficiency, this group was fed a zinc-deficient diet. Group 2: In addition to maternal zinc deficiency, this group was fed a standard diet. Group 3: In addition to maternal zinc deficiency, this group was fed a standard diet and received additional zinc supplementation. Group 4: Control group. Pancreas ZnT8 levels were determined by ELISA method and insulin-positive cell ratios in β-cells by immunohistochemistry.ResultsThe highest pancreatic ZnT8 levels and anti-insulin positive cell ratios in the current study were obtained in Group 3 and Group 4. In our study, the lowest pancreatic ZnT8 levels were obtained in Group 1 and Group 2, and the lowest pancreatic anti-insulin positive cell ratios were obtained in Group 1.ConclusionThe results of the present study; in rats fed a zinc-deficient diet after maternal zinc deficiency has been established shows that ZnT8 levels and anti-insulin positive cell ratios in pancreatic tissue, which is significantly suppressed, reach control values with intraperitoneal zinc supplementation.     

Studies on intracellular transport of secretory proteins in the rat exocrine pancreas

Summary Prolonged secretory stimulation of the exocrine pancreas in the rat by in vivo infusion of caerulein leads to a rapid degranulation of the organ associated with a progressive reduction in the size of the zymogen granules. During the first six to twelve hours of stimulation Golgi complexes are enlarged and several structural forms of multivesicular bodies are found indicating a lysosomal degradation of membrane material in the Golgi area. Maximum secretory activity is obtained after a 24 hour infusion, Golgi complexes appear fragmented, the secretory granules measure only 1/3 to 1/4 their normal size. Thereafter, in spite of a continuous stimulation, the exocrine cells regranulate progressively up to 72 hours of infusion. This regranulation is associated with massive enlargement of the Golgi complexes.The phasic adaptation of the exocrine pancreas to prolonged stimulation, concluded from the structural studies, was confirmed by biochemical analysis of protein synthesis, intracellular transport and enzyme discharge. Pancreatic protein synthesis as measured by the incorporation of tritiated leucine remained unchanged during the first six hours of stimulation, then increased reaching a maximum of 230% of the control levels after 24 hours of infusion. After 48 and 72 hours the rate of protein synthesis decreased again to normal values. Most pronounced changes were observed in the kinetics of intracellular transport of newly synthesized proteins. Using pulse-chase incubation of prestimulated pancreatic lobules, the rate of transition of secretory proteins through the cell increased consistently with prolonged infusion periods reaching maximal acceleration after 24 hours. Newly synthesized proteins were transported and segregated up to ten times faster than in controls. After a maximum at 24 hours transport returned to normal rates after 72 hours of infusion. Enzyme secretion, measured for amylase, followed a similar pattern of stimulation.The results suggest a phasic adaptation of the exocrine pancreatic cell to prolonged stimulation. They demonstrate for the first time the possibility of an acceleration of intracellular transport by means of secretagogues.Dedicated to Professor W. Bargmann on the occasion of his 70th birthday.Supported by a grant from Deutsche Forschungsgemeinschaft (Ke 113/8). A preliminary communication was presented at the 9th annual meeting of the European Society for Clinical Investigation, Rotterdam (April 24–26, 1975). The expert technical assistance of Miss Helga Hollerbach and Miss Hiltraud Hosser is gratefully acknowledged.     

Regulatory effects of insulin and experimental diabetes on neutral amino acid transport in the perfused rat exocrine pancreas. Kinetics of unidirectional l-serine influx and efflux at the basolateral plasma membrane

Relatively little is known about the hormonal regulation of amino acid transport in the normal and diabetic exocrine pancreas. In this study unidirectional influx and tracer efflux of l-serine at the basolateral interface of the rat pancreatic epithelium was investigated in the perfused exocrine pancreas using a rapid (< 30 s) paired-tracer dilution technique. In the non-diabetic pancreas l-serine influx was saturable and stimulated by perfusion with exogenous bovine insulin (100 μU/ml). Transport of l-serine and methylaminoisobutyric acid was markedly elevated in pancreata isolated from streptozotocin diabetic rats and insulin partially reversed the stimulation of l-serine transport induced by experimental diabetes. These results suggest that insulin and diabetes modulate the epithelial transport activity for small neutral amino acids in the intact exocrine pancreas.     

Constitutive protein secretion from the exocrine pancreas of fetal rats

Two general kinds of exocytotic secretion of proteins are known: that which is stimulated by secretagogues; and constitutive exocytosis, which is unable to be stimulated. The exocrine pancreas has often been cited as a model system for the first kind of secretion. However, the release of digestive enzymes from the exocrine pancreas of 1-day prenatal rats cannot be stimulated by secretagogues; therefore, its secretion is constitutive. To gain insight into the intracellular pathways which mediate secretion in the fetal gland, we examined the kinetics of release of newly synthesized proteins. We find that fetal pancreas in a steady state of secretion releases pulse-labeled secretory proteins in two kinetically distinct phases. The first phase occurring during 0-6.5 h of chase comprises approximately 12% of total incorporated radioactivity, the second phase beginning at greater than 7 h of chase comprises the remainder. Based on analysis by electron microscope autoradiography, radiolabel is localized during the first phase of secretion in immature granules/condensing vacuoles, Golgi compartments, and few mature granules. The second phase of secretion occurs when radiolabel is predominantly in mature granules. We propose that secretion occurs via (at least) 2 exocytotic routes, both of which are constitutive in fetal pancreatic tissue.     

Pancreatic regeneration after chronic ethanol feeding in rats.

Increase of phosphatidic acid (PA) accumulation in response to caerulein (Cae) and after subtotal pancreatectomy (SP) has been previously described and phospholipase D (PLD) derived PA involvement in pancreatic regeneration was suggested. We also described decrease of Cae stimulated PA accumulation after a single dose of ethanol (both in vitro and in vivo). The present study was undertaken in order to determine the influence of chronic ethanol feeding on basal and Cae stimulated PA accumulation and other parameters of pancreatic regeneration in control and ethanol feed rats. Male rats were pair fed ad libitum with an isocaloric liquid diet (Lieber De Carli) with or without ethanol. In vitro study: pair fed rats were killed after 2 or 6 weeks of feeding, pancreata were dissected out and weighted, dispersed pancreatic acini were then prepared and loaded with 3H myristic acid in order to label the phosphatidylcholine pool. Phosphatidic acid (3H PA) accumulation, in the presence of propranolol, was measured after stimulation with increasing doses of Cae. In vivo study: PA was measured 3 days after SP or sham operation in both groups of rats, and also after 1 h of Cae infusion (0.25 microg/kg/h). Pancreatic weight, amylase, protein, RNA and DNA content were established. Results: In vitro study 1) Basal PLD activity expressed as PA accumulation was significantly elevated after 6 weeks of ethanol feeding in comparison to the control values (28%). 2) Cae in doses ranging from 100 pM to 5 nM was not able to stimulate PA accumulation in isolated pancreatic acini prepared from ethanol fed rats. In vivo study: 1) Body weight and pancreatic weight were similar in, both the ethanol fed and the control groups, after 2 and 6 weeks. 2) Ethanol feeding significantly decreased total amylase content in the pancreas, but did not change protein, RNA and DNA content. 3) in contrast to the control animals in which SP caused a 71.1% increase of PA accumulation over the sham operation, subtotal pancreatectomy was not able to stimulate PA in rats fed with ethanol. 4) Also Cae infusion did not stimulate PA accumulation in the ethanol fed animals in comparison to the controls. Since the involvement of PLD activation in the early stages of pancreatic regeneration was postulated, our results suggest that chronic ethanol feeding can influence this process by decrease of PA production, probably because of the inhibition of hydrolytic PLD activity in the presence of ethanol. This could be one of the mechanisms responsible for pancreatic injury after chronic ethanol consumption.     

Substrate specificity in the control of digestive enzymes in larvae of the black carpet beetle

When starved larvae of the black carpet beetle, Attagenus megatoma, were fed selected diets, increases in proteolytic, trypsin, and chymotrypsin activity were correlated with total midgut protein and not with the amount of food consumed. Although larvae initially consumed more of a starch diet than of 2 diets that contained added protein, total protease activity in these larvae was minimal. Starch-fed larvae and larvae fed a casein-sucrose diet had a consistently higher level of sucrase activity than larvae fed an all-casein diet. These total results support a secretagogue mechanism for control of digestive enzyme synthesis in insects. In addition, the absence of parallel stimulation of different digestive enzymes by a single substrate (starch) indicated nutrient class specificity in the control of inducible midgut enzymes in this species.     

High density and food deprivation affect arginine vasotocin, isotocin and melatonin in gilthead sea bream (Sparus auratus).

Arginine vasotocin (AVT) and isotocin (IT) levels in plasma and pituitary, and melatonin (MEL) levels in plasma were determined in gilthead sea bream (Sparus auratus) subjected to two different types of stress: i) high density (HD) and ii) food deprivation (NF: non-fed). Fishes were randomly assigned to one of 4 treatments that lasted for 14 days: 1) fed fish under normal low density (ND, 4 kg m(-3)); 2) non-fed (NF) fish under ND; 3) fed fish under high density (HD, 70 kg m(-3)); and 4) non-fed fish under HD. Ten fish from each tank were anaesthetized, weighed and plasma and pituitary samples were taken. Plasma and pituitary AVT and IT content were determined by HPLC, while plasma MEL was assayed by RIA. Plasma AVT and IT values were enhanced in all fish kept at high density. The response of AVT was much stronger than that of IT. The highest pituitary AVT and IT levels were shown in NF fish kept at normal density. The significantly higher plasma MEL levels were measured in fed fish kept at HD. These results suggest a role of AVT, IT and MEL in response of sea bream to a common stress factor, high density. Although food deprivation does not influence AVT and IT plasma levels, it seems to affect hypothalamic synthesis of nonapeptides. Further studies are required to elucidate the complex role of AVT, IT and MEL in the sea bream's response to different stress stimuli.