Molecular mechanisms of alcohol associated pancreatitis

Alcohol abuse is commonly associated with the development of both acute and chronic pancreatitis. Despite this close association, the fact that only a small percentage of human beings who abuse alcohol develop pancreatitis indicates that alcohol abuse alone is not sufficient to initiate clinical pancreatitis. This contention is further supported by the fact that administration of ethanol to experimental animals does not cause pancreatitis. Because of these findings, it is widely believed that ethanol sensitizes the pancreas to injury and additional factors trigger the development of overt pancreatitis. How ethanol sensitizes the pancreas to pancreatitis is not entirely known. Numerous studies have demonstrated that ethanol and its metabolites have a number of deleterious effects on acinar cells. Important acinar cells properties that are affected by ethanol include: calcium signaling, secretion of zymogens, autophagy, cellular regeneration, the unfolded protein response, and mitochondrial membrane integrity. In addition to the actions of ethanol on acinar cells, it is apparent that ethanol also affects pancreatic stellatecells. Pancreatic stellate cells have a critical role in normal tissue repair and the pathologic fibrotic response. Given that ethanol and its metabolites affect so many pancreatic functions, and that all of these effects occur simultaneously, it is likely that none of these effects is "THE" effect. Instead, it is most likely that the cumulative effect of ethanol on the pancreas predisposes the organ to pancreatitis. The focus of this article is to highlight some of the important mechanisms by which ethanol alters pancreatic functions and may predispose the pancreas to disease.     

Cholinergic Mediation of Alcohol‐Induced Experimental Pancreatitis

Objectives: The mechanisms initiating pancreatitis in patients with chronic alcohol abuse are poorly understood. Although alcohol feeding has been previously suggested to alter cholinergic pathways, the effects of these cholinergic alterations in promoting pancreatitis have not been characterized. For this study, we determined the role of the cholinergic system in ethanol‐induced sensitizing effects on cerulein pancreatitis. Methods: Rats were pair‐fed control and ethanol‐containing Lieber‐DeCarli diets for 6 weeks followed by parenteral administration of 4 hourly intraperitoneal injections of the cholecystokinin analog, cerulein at 0.5 μg/kg. This dose of cerulein was selected because it caused pancreatic injury in ethanol‐fed but not in control‐fed rats. Pancreatitis was preceded by treatment with the muscarinic receptor antagonist atropine or by bilateral subdiaphragmatic vagotomy. Measurement of pancreatic pathology included serum lipase activity, pancreatic trypsin, and caspase‐3 activities, and markers of pancreatic necrosis, apoptosis, and autophagy. In addition, we measured the effects of ethanol feeding on pancreatic acetylcholinesterase activity and pancreatic levels of the muscarinic acetylcholine receptors m1 and m3. Finally, we examined the synergistic effects of ethanol and carbachol on inducing acinar cell damage. Results: We found that atropine blocked almost completely pancreatic pathology caused by cerulein administration in ethanol‐fed rats, while vagotomy was less effective. Ethanol feeding did not alter expression levels of cholinergic muscarinic receptors in the pancreas but significantly decreased pancreatic acetylcholinesterase activity, suggesting that acetylcholine levels and cholinergic input within the pancreas can be higher in ethanol‐fed rats. We further found that ethanol treatment of pancreatic acinar cells augmented pancreatic injury responses caused by the cholinergic agonist, carbachol. Conclusion: These results demonstrate key roles for the cholinergic system in the mechanisms of alcoholic pancreatitis.     

Animal and in vitro models of alcoholic pancreatitis: role of cholecystokinin

Although ethanol abuse is the major etiologic factor in the development of acute and chronic pancreatitis, the mechanisms of ethanol effects to cause pancreatitis are poorly understood. The major reason for the lack of progress is the relative lack of animal models that reproduce the deleterious effects of ethanol on the pancreas that are observed in human disease. We propose that the effect of ethanol on the pancreas is due to its ability to sensitize animals and humans to the potentially injurious effects of other stimuli. We have developed models of ethanol-induced acute and chronic pancreatitis in rats as well as pancreatic acinar cells in primary culture demonstrating that ethanol sensitizes the pancreas to the inflammatory, cell death, and fibrosing responses caused by cholecystokinin (CCK). Our results indicate that the ethanol-sensitized inflammatory response is the key or trigger event for the development of the other pathologic responses in both acute and chronic pancreatitis, such as cell death, intracellular digestive enzyme activation, and fibrosis. These findings suggest that experimental strategies designed to reveal the modulating effects of ethanol on the mechanisms underlying the inflammatory, cell death, and fibrosing responses stimulated by CCK will provide the key information needed to understand how ethanol abuse causes pancreatitis.     

Chronic ethanol consumption increases the fragility of rat pancreatic zymogen granules.

Intracellular activation of pancreatic digestive enzymes by lysosomal hydrolases is thought to be an early event in the pathogenesis of pancreatic injury. As ethanol excess is an important association of pancreatitis, experimental work has been directed towards exploring possible mechanisms whereby ethanol may facilitate contact between inactive digestive enzyme precursors and lysosomal enzymes. The aim of this study was to find out if chronic ethanol administration increases the fragility of rat pancreatic zymogen granules. Sixteen male Sprague-Dawley rats were pair fed ethanol and control liquid diets for four weeks. Zymogen granule fragility was then assessed in pancreatic homogenate by determination of (a) latency and (b) per cent supernatant enzyme after sedimentation of zymogen granules. Amylase was used as a zymogen granule marker enzyme. Latency was significantly reduced in pancreatic homogenates of ethanol fed animals suggesting increased zymogen granule fragility. In support of this finding, there was a trend towards increased supernatant enzyme after ethanol feeding. In conclusion, administration of ethanol increases the fragility of pancreatic zymogen granules in the absence of morphological evidence of pancreatic injury. It is proposed that zymogen granule fragility may play an early part in the pathogenesis of alcoholic pancreatitis by permitting contact between digestive and lysosomal enzymes.     

Animal Models in Alcoholic Pancreatitis — What Can We Learn?

Although the majority of patients with chronic pancreatitis present a history of excessive alcohol consumption, the pathophysiology underlying chronic alcoholic pancreatitis remains poorly defined. Since experimental animal models represent helpful tools in understanding human disease, numerous laboratory studies have been designed to study the effects of alcohol on the pancreas. In the present article we summarize the existing animal models that have been used to investigate the effects of acute and chronic alcohol application on the development of morphological alterations and pancreatic injury. Despite considerable experimental effort, acute or chronic ethanol feeding alone failed to cause acute or chronic pancreatitis in animals. However, ethanol-feeding and the combination with other procedures has demonstrated several mechanisms that play a role in ethanol-induced pancreatic injury. Among these ethanolinduced alterations and mechanisms are the reduction of pancreatic blood-flow and microcirculation, damaging effects of ethanol metabolites, increased pancreatic acinar cell expression of digestive and lysosomal enzymes, increased glandular enzyme content, additional nutritional factors, pancreatic duct obstruction, and limitations of pancreatic regeneration. Although no satisfactory animal model for alcoholic pancreatitis has been developed, these animal models have provided insights in several factors that predispose the pancreas to development of pancreatic injury and contribute to alcoholic pancreatitis.     

Effects of ethanol and protein deficiency on pancreatic digestive and lysosomal enzymes.

The pathogenesis of alcoholic pancreatitis is not fully understood. An increase in pancreatic digestive and lysosomal enzyme synthesis because of ethanol consumption could contribute to the development of pancreatic injury in alcoholics. This study aimed, firstly, to determine the effect of ethanol on the content and messenger RNA levels of pancreatic digestive enzymes and on the messenger RNA level of the lysosomal enzyme cathepsin B, and secondly, to examine the influence of concomitant protein deficiency (a known association of alcoholism and pancreatic injury) on these effects. A rat model of chronic ethanol administration was used in which rats were fed in groups of four, and for four weeks, protein sufficient and protein deficient diets with or without ethanol. Ethanol increased the pancreatic content of lipase but did not influence chymotrypsinogen or trypsinogen values. mRNA levels for lipase, trypsinogen, and chymotrypsinogen were raised in rats fed ethanol. Protein deficiency resulted in reduced tissue levels of lipase, chymotrypsinogen, and amylase but did not influence trypsinogen values. mRNA levels for proteases were increased in protein deficient rats, while those for lipase remained unaltered. Both ethanol and protein deficiency increased mRNA levels for cathepsin B. It is concluded that chronic ethanol consumption, in both protein sufficient and protein deficient states, increases the capacity of the pancreatic acinar cell to synthesise digestive and lysosomal enzymes.     

Alcohol abuse, endoplasmic reticulum stress and pancreatitis

Alcohol abuse is a common cause of both acute and chronic pancreatitis. There is a wide spectrum of pancreatic manifestations in heavy drinkers from no apparent disease in most individuals to acute inflammatory and necrotizing pancreatitis in a minority of individuals with some progressing to chronic pancreatitis characterized by replacement of the gland by fibrosis and chronic inflammation. Both smoking and African-American ethnicity are associated with increased risk of alcoholic pancreatitis. In this review we describe how our recent studies demonstrate that ethanol feeding in rodents causes oxidative stress in the endoplasmic reticulum (ER) of the digestive enzyme synthesizing acinar cell of the exocrine pancreas. This ER stress is attenuated by a robust unfolded protein response (UPR) involving X-box binding protein-1 (XBP1) in the acinar cell. When the UPR activation is prevented by genetic reduction in XBP1, ethanol feeding causes significant pathological responses in the pancreas. These results suggest that the reason most individuals who drink alcohol heavily do not get significant pancreatic disease is because the pancreas mounts an adaptive UPR to attenuate the ER stress that ethanol causes. We hypothesize that disease in the pancreas results when the UPR is insufficiently robust to alleviate the ER stress caused by alcohol abuse.     

Emerging concepts for the mechanism of alcoholic pancreatitis from experimental models

The pathophysiologic mechanisms that underlie acute and chronic pancreatitis arising from alcohol abuse are poorly understood. The reasons for this state of knowledge result historically from a lack of models for experimental investigation. Ethanol feeding alone, even at high doses, has minimal and inconsistent effects on morphologic findings in the pancreas in experimental animals. This experience, plus the fact that alcohol abuse causes pancreatic pathology in only a minority of patients, suggest that ethanol acts to sensitize the pancreas to the deleterious effects of other stimuli. In this article, we discuss findings to support this concept of ethanol as a sensitizing agent and experimental models developed that can be used to investigate the effects of ethanol on the pathologic processes of pancreatitis. These pathologic processes include inflammation, cell death, intrapancreatic digestive enzyme activation, and fibrosis.     

Pathobiology of alcoholic pancreatitis.

This paper provides a summary of the effects of alcohol abuse on the pathobiologic responses that occur during acute and chronic pancreatitis considering both the human disease and animal/tissue models. The effects are multiple and include ones on cell death leading to necrosis; on inflammation resulting in a sensitized response to pancreatic stress; and fibrosis through effects of ethanol on pancreatic stellate cells and the plasminogen system. Although the effects of alcohol are multiple and complex, it is likely that a combination of a few key effects on these pathobiologic responses drive the increased sensitivity of the pancreas to acute pancreatitis with pancreatic stress and the promotion of chronic pancreatitis with pancreatic injury occurring during acute pancreatitis.     

Mechanisms of alcoholic pancreatitis

Alcoholic pancreatitis is a major complication of alcohol abuse. The risk of developing pancreatitis increases with increasing doses of alcohol, suggesting that alcohol exerts dose-related toxic effects on the pancreas. However, it is also clear that only a minority of alcoholics develop the disease, indicating that an additional trigger may be required to initiate clinically evident pancreatic injury. It is now well established that alcohol is metabolized by the pancreas via both oxidative and non-oxidative metabolites. Alcohol and its metabolites produce changes in the acinar cells, which may promote premature intracellular digestive enzyme activation thereby predisposing the gland to autodigestive injury. Pancreatic stellate cells (PSCs) are activated directly by alcohol and its metabolites and also by cytokines and growth factors released during alcohol-induced pancreatic necroinflammation. Activated PSCs are the key cells responsible for producing the fibrosis of alcoholic chronic pancreatitis. Efforts to identify clinically relevant factors that may explain the susceptibility of some alcoholics to pancreatitis have been underway for several years. An unequivocal, functionally characterized, association is yet to be identified in clinical studies, although in the experimental setting, endotoxin has been shown to trigger overt pancreatic injury and to promote disease progression in alcohol-fed animals. Thus, while the molecular effects of alcohol on the pancreas have been increasingly clarified in recent years, identification of predisposing or triggering factors remains a challenge.     

Protease-activated receptor-2 protects against pancreatitis by stimulating exocrine secretion

BACKGROUND: Protease-activated receptor-2 (PAR-2) is present in the pancreas, where it has been shown to play a protective role during pancreatitis. However, the mechanism by which it protects against pancreatitis still remains to be elucidated. Acute pancreatitis is associated with premature zymogen activation and a blockage in digestive enzyme secretion. AIM: To examine the effects of PAR-2 activation on the severity of pancreatitis, and to determine whether its protective effects are mediated by affecting either premature activation or secretory blockage, or both. RESULTS: The results confirmed that PAR-2 -/- mice have more severe pancreatitis than wild-type mice. Interestingly, intrapancreatic trypsin levels in the PAR-2 knockouts remained high after 6 h of pancreatitis, whereas they reverted to normal in the wild types. During pancreatitis, PAR-2 mRNA levels were upregulated in wild-type mice in response to supramaximal caerulein administration. Further, after a single injection of supramaximal caerulein, PAR-2 mRNA levels were also elevated, reaching a peak at 3 h. Stimulating PAR-2 with trypsin or the PAR-2-activating peptide, serine-leucine-isoleucine-glycine-arginine-leucine (SLIGRL), induced significantly more secretion from the acini of these caerulein-sensitised mice compared with the controls. PAR-2 activation also reversed the inhibition of secretion observed in both the caerulein and arginine models. CONCLUSIONS: Trypsin released during the early stages of pancreatitis activates PAR-2 receptors on the acinar cells and stimulates secretion from these cells. Thus, PAR-2 activation may decrease pancreatic injury and limit the severity of pancreatitis by allowing extracellular trypsin to act as a secretagogue.     

Effects of short-term pancreatic duct obstruction in rats.

The short-term effects of rat pancreatic duct obstruction were evaluated and compared with those recently reported to follow obstruction of the rabbit pancreatic duct. In both species pancreatic edema and hyperamylasemia are noted, and the lysosomal hydrolase cathepsin B is redistributed from the lysosome-enriched to the zymogen granule-enriched subcellular fraction. Theoretically, this redistribution phenomenon might lead to digestive enzyme activation because cathepsin B is known to be capable of activating trypsinogen. The hyperamylasemia and pancreatic edema (but not the cathepsin B redistribution) that follow rat pancreatic duct obstruction were increased by infusion of a submaximally stimulating dose of the cholecystokinin analogue cerulein. Administration of the cholecystokinin-receptor antagonist L-364,718 reduced the hyperamylasemia but did not alter the pancreatic edema or cathepsin B redistribution. These observations indicate that cholecystokinin may modulate some but not all of the effects of duct obstruction. Secretin administration increased the degree of pancreatic edema and had no demonstrable protective effect. The rat duct-obstruction model described in this report may prove particularly useful in future studies designed to clarify the early events underlying the development of acute pancreatitis.     

Pathomorphological changes in microcirculation of pancreas during experimental acute pancreatitis

BACKGROUND/AIMS: Severe acute pancreatitis is prevalent in Eastern Bohemia (a part of the Czech Republic) and remains a very difficult problem to manage. Recent studies in treatment there are quite frequent but a direct view into the pancreas during its inflammatory process is very rare. Only information about a normal pancreatic microvascular bed appears to be available. This study was designed to explore pathomorphological changes in pancreatic microcirculation at the start and during the development of acute pancreatitis. METHODOLOGY: A group of 50 laboratory white rats was studied. The acute pancreatitis was induced by the modified method of Siech et al. The method of clamping of biliopancreatic duct and stimulation of external secretory tissue by a cholecystokinin and secretin and oral (orogastric tube) ethanol administration was performed. The pancreatic microvascular patterns were observed by using histochemical and corrosion casts methods. RESULTS: The study of the corrosion casts of pancreatic microcirculation in the scanning electron microscope and histochemical studies demonstrated the visible reduction of the pancreatic microvascular bed 18 hours after induction of acute pancreatitis. The microvascular bed is not fully destroyed until 48 hours of acute pancreatitis. CONCLUSIONS: The model of acute pancreatitis using postoperative application of ethanol to the digestive tract after stimulation of pancreas by cholecystokinine and secretin in the rats seems to be real and useful. The study of the corrosion casts of microcirculation in the scanning electron microscope and histochemical studies demonstrated the visible reduction of the pancreatic microvascular bed 18 hours after induction of acute pancreatitis. The microvascular bed is not fully destroyed until 48 hours of running acute pancreatitis, as some "islets" of the vital tissue still have undestroyed microvessels at this time. Despite the above-mentioned serious changes, restricted pancreatic microcirculation enables blood and medicament distribution to the still intact pancreatic tissue.     

Pathophysiology of alcohol-induced pancreatitis

Excessive ethanol consumption is a common risk factor for acute and chronic pancreatitis. Ethanol could lead to the onset of pancreatitis in a number of ways; the most recently discovered is its effect on intrapancreatic digestive enzyme activation, by either sensitizing acinar cells to pathologic stimuli or stimulating the release of a secretagogue (cholecystokinin) from duodenal I cells. Recent advances in cell biologic and molecular techniques have permitted us to address the intracellular events involved in digestive enzyme activation in a manner that was previously considered impossible. Investigations that used these novel techniques found that (a) trypsin is, in contrast to its role in the small intestine, not necessarily involved in the premature intracellular activation of other digestive proteases such as proelastase; (b) trypsinogen does not autoactivate intracellularly but is instead largely activated by the lysosomal hydrolase cathepsin B; and (c) the role of trypsin in the intrapancreatic protease cascade is most likely one that involves the degradation, rather than the activation, of active digestive proteases including trypsin itself. These studies, as well as investigations that have addressed the role of mutant trypsin in the disease onset of hereditary pancreatitis, suggest that trypsin may not be critical for triggering pancreatitis but might have a protective role against the action of some of the other digestive proteases. While the specific role of different digestive enzymes in initiating pancreatitis is still a matter of debate and the topic of ongoing investigations, experimental evidence suggests that ethanol can directly interfere with the processes involved in digestive zymogen activation.     

Experimental pancreatitis is mediated by low-affinity cholecystokinin receptors that inhibit digestive enzyme secretion.

Rats infused with a supramaximally stimulating dose of the cholecystokinin (CCK) analog caerulein develop acute edematous pancreatitis. Using CCK-JMV-180, a recently developed CCK analog that acts as an agonist at high-affinity CCK receptors but antagonizes the effect of CCK at low-affinity receptors, we have determined that caerulein induces pancreatitis by interacting with low-affinity CCK receptors. Those low-affinity receptors mediate CCK-induced inhibition of digestive enzyme secretion from the pancreas. Our observations, therefore, suggest that this form of experimental pancreatitis results from the inhibition of pancreatic digestive enzyme secretion.     

A proinflammatory, antiapoptotic phenotype underlies the susceptibility to acute pancreatitis in cystic fibrosis transmembrane regulator (-/-) mice

BACKGROUND & AIMS: Cystic fibrosis transmembrane regulator (CFTR) gene mutations are associated with pancreatic insufficiency and pancreatitis. Chronic pancreatitis, including cystic fibrosis-related disease, may exist as a continuum between acute and chronic disease and may manifest as recurrent pain. We hypothesized that cftr(m1UNC) (-/-) mice, which have no evidence of chronic pancreatitis, are susceptible to developing acute pancreatitis. METHODS: We used a cerulein hyperstimulation model of acute pancreatitis and measured histological changes, tissue edema, neutrophil infiltration, inflammatory mediators' mRNA expression, apoptosis markers, and pancreatic trypsin and serum lipase activities. Additionally, we quantitated in vivo pancreatic secretion and pancreatic digestive enzymes. RESULTS: Multiple proinflammatory cytokine genes were constitutively overexpressed in cftr (-/-) pancreas compared with wild-type mice. During acute pancreatitis, cftr (-/-) mice developed more severe acute pancreatitis than wild-type, as indicated by greater pancreatic edema, neutrophil infiltration, mRNA expression of multiple inflammatory mediators, and less apoptotic cell death. In contrast to wild-type mice, cftr (-/-) mice had blunted increases in pancreatic trypsin and serum lipase activities, but similar percentages of pancreatic trypsinogen activation. Finally, cftr (-/-) mice had less in vivo pancreatic secretion in response to cholecystokinin octapeptide and reduced pancreatic digestive enzyme protein and mRNA levels, thus suggesting mild pancreatic insufficiency. CONCLUSIONS: A baseline proinflammatory state and an antiapoptotic phenotype may sensitize cftr (-/-) mice to developing more severe acute pancreatitis with an exuberant pancreatic inflammatory response. Cftr (-/-) mice have mild pancreatic insufficiency, which partially explains the blunted increase of pancreatic and serum digestive enzymes during acute pancreatitis. These findings may explain the susceptibility to acute pancreatitis in persons with classic and nonclassic cystic fibrosis.     

Interactive effects of dietary protein and ethanol on rat pancreas. Protein synthesis and enzyme secretion

Nutritional factors, especially the protein and fat content of the diet, may alter the likelihood of pancreatic injury after a number of insults, including chronic ethanol intake. This issue was studied experimentally by match-feeding rats liquid diets of varying protein content with and without ethanol. Protein synthesis and enzyme secretion were investigated, because these parameters are believed to increase the capacity for pancreatic autodigestion. Protein synthesis was assessed by determining the incorporation of tritiated phenylalanine into trichloroacetic acid precipitated protein 10 minutes after IP injection and then corrected for the size of the precursor pool. Enzyme secretion was studied using pancreatic acini, which were prepared using clostripain-poor collagenase. Chronic ethanol feeding stimulated protein synthesis and lipase secretion and content in rats receiving adequate amounts of protein. These stimulatory effects of ethanol were markedly attenuated in rats administered protein poor diets. Protein deficiency per se significantly decreased the weight, protein, and enzyme content of the rat pancreas as well as increased the percentage release of lipase from acini. Although extrapolation from animal studies may be tenuous, the present findings may explain the link between nutrition and the occurrence of alcoholic pancreatitis.     

Both thermal and non-thermal stress protect against caerulein induced pancreatitis and prevent trypsinogen activation in the pancreas.

BACKGROUND AND AIM: Recent studies have indicated that prior thermal stress causes upregulation of heat shock protein 70 (HSP70) expression in the pancreas and protects against secretagogue induced pancreatitis. The mechanisms responsible for the protective effect are not known. Similarly, the effects of prior non-thermal stress on HSP70 expression and pancreatitis are not known. The current studies were designed to specifically address these issues. METHODS: In the current studies pancreatitis was induced by administration of a supramaximally stimulating dose of caerulein 12 hours after thermal stress and 24 hours after non-thermal (that is, beta adrenergic stimulation) stress. RESULTS: Both thermal and non-thermal stresses caused pancreatic HSP70 levels to rise and resulted in increased expression of HSP70 in acinar cells. Both forms of stresses protected against caerulein induced pancreatitis and prevented the early intrapancreatic activation of trypsinogen which occurs in this model of pancreatitis. CONCLUSIONS: These results suggest that both thermal and non-thermal stresses protect against pancreatitis by preventing intrapancreatic digestive enzyme activation and that HSP70 may mediate this protective effect.     

Pancreatic acinar cell function and morphology in rats chronically fed an ethanol diet

The aim of the present study was to determine the effect of prolonged ethanol intake on the morphology and protein metabolism in the rat pancreatic acinar cells. Weight-matched triplets of Sprague-Dawley rats were fed Lieber-DeCarli diet containing 5% (wt/vol) concentration of ethanol, isocaloric amounts of Lieber-DeCarli diet, or rat chow ad libitum for 6, 12, and 18 mo. In the ethanol-fed group, histologic studies by light microscopy showed absence of protein plugs in the pancreatic ducts and/or pancreatitis, but electron-microscopic evaluation revealed progressive accumulation of lipid droplets in acinar and ductal cells. No definite changes in the mitochondria and endoplasmic reticulum were noticed. Biochemical studies revealed increased specific activity of trypsinogen, chymotrypsinogen, and lipase, and decreased specific activity of amylase. Trypsin-inhibiting capacity was decreased in the tissue and in the medium in a progressive fashion. There was no increase in the secretion of total protein. These data show a complex and a nonparallel alteration of specific digestive enzymes and trypsin inhibitor in this model of chronic ethanol intoxication that may be of relevance to occurrence of pancreatitis.