Vijay P. Singh

Phosphatidylinositol 3-kinase-dependent activation of trypsinogen modulates the severity of acute pancreatitis

Intra-acinar cell activation of digestive enzyme zymogens including trypsinogen is generally believed to be an early and critical event in acute pancreatitis. We have found that the phosphatidylinositol 3-kinase inhibitor wortmannin can reduce the intrapancreatic activation of trypsinogen that occurs during two dissimilar experimental models of rodent acute pancreatitis, secretagogue- and duct injection-induced pancreatitis. The severity of both models was also reduced by wortmannin administration. In contrast, the NF-kappa B activation that occurs during the early stages of secretagogue-induced pancreatitis is not altered by administration of wortmannin. Ex vivo, caerulein-induced trypsinogen activation is inhibited by wortmannin and LY294002. However, the cytoskeletal changes induced by caerulein were not affected by wortmannin. Concentrations of caerulein that induced ex vivo trypsinogen activation do not significantly increase phosphatidylinositol-3,4-bisphosphate or phosphatidylinositol 3,4,5-trisphosphate levels or induce phosphorylation of Akt/PKB, suggesting that class I phosphatidylinositol 3-kinases are not involved. The concentration of wortmannin that inhibits trypsinogen activation causes a 75% decrease in phosphatidylinositol 3-phosphate, which is implicated in vesicle trafficking and fusion. We conclude that a wortmannin-inhibitable phosphatidylinositol 3-kinase is necessary for intrapancreatic activation of trypsinogen and regulating the severity of acute pancreatitis. Our observations suggest that phosphatidylinositol 3-kinase inhibition might be of benefit in preventing acute pancreatitis.

Heat shock protein 70 prevents secretagogue-induced cell injury in the pancreas by preventing intracellular trypsinogen activation.

Rodents given a supramaximally stimulating dose of cholecystokinin or its analogue cerulein develop acute pancreatitis with acinar cell injury, pancreatic inflammation, and intrapancreatic digestive enzyme (i.e., trypsinogen) activation. Prior thermal stress is associated with heat shock protein 70 (HSP70) expression and protection against cerulein-induced pancreatitis. However, thermal stress can also induce expression of other HSPs. The current studies were performed using an in vitro system to determine whether HSP70 can actually mediate protection against pancreatitis and, if so, to define the mechanism underlying that protection. We show that in vitro exposure of freshly prepared rat pancreas fragments to a supramaximally stimulating dose of cerulein results in changes similar to those noted in cerulein-induced pancreatitis, i.e., intra-acinar cell trypsinogen activation and acinar cell injury. Short-term culture of the fragments results in HSP70 expression and loss of the pancreatitis-like changes noted after addition of cerulein. The culture-induced enhanced HSP70 expression can be prevented by addition of either the flavonoid antioxidant quercetin or an antisense oligonucleotide to HSP70. Under these latter conditions, addition of a supramaximally stimulating concentration of cerulein results in trypsinogen activation and acinar cell injury. These findings indicate that the protection against cerulein-induced pancreatitis that follows culture-induced (and possibly thermal) stress is mediated by HSP70. They suggest that the HSP acts by preventing trypsinogen activation within acinar cells.

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, β 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.

Elevated Serum Triglycerides are Independently Associated With Persistent Organ Failure in Acute Pancreatitis

Objectives:Hypertriglyceridemia (HTG) represents a major health problem with prevalence exceeding 30% in the U.S. The present study aims to assess the effect of elevated serum triglyceride (TG) levels on the severity of acute pancreatitis (AP).Methods:Prospectively enrolled AP patients were categorized into normal, mild, moderate, and severe/very severe categories based on their TG levels and compared in respect to demographics, comorbidities, and clinical outcomes. Multivariate analysis determined whether elevated TG levels were independently associated with persistent organ failure.Results:Two hundred and one out of 400 AP patients had serum TGs measured within 72 h of presentation, of which 115 had normal TG levels and 86 HTG (20 mild, 41 moderate, and 25 severe/very severe). Patients with HTG were of younger age (44 vs. 52 years), predominantly male (65% vs. 45%), obese (57% vs. 34%), diabetic (38% vs. 17%), and developed more frequently persistent organ failure (40% vs. 17%) compared with those with normal TGs (P<0.02). The rate of persistent organ failure increased proportionally with HTG severity grades (17% when normal TGs, 30% in mild, 39% in moderate, and 48% in severe/very severe HTG, Ptrend<0.001). On multivariate analysis controlling for age, gender, body mass index, diabetes, and alcohol etiology, moderate HTG (odds ratio (OR), 2.6; P=0.04) and severe/very severe HTG (OR, 4.9; P=0.009) were independently associated with persistent organ failure.Conclusions:Elevated serum TGs in AP patients are independently and proportionally correlated with persistent organ failure regardless of etiology. TG-mediated lipotoxicity may be an attractive target to design novel interventions for severe AP.

Patients With Sentinel Acute Pancreatitis of Alcoholic Etiology Are at Risk for Organ Failure and Pancreatic Necrosis: A Dual-Center Experience.

Objectives To assess the relationship between alcoholic etiology, tobacco use, and severe acute pancreatitis (AP). Methods Smoking and alcohol exposure were recorded upon admission in a cohort of patients with AP within the United States. Patients with first, “sentinel” attack of AP were identified for analysis. Associations between alcohol, smoking, and severe AP were validated in an independent cohort of patients from Spain. Results US cohort (n = 222): Thirty-five percent developed organ failure (OF), 35% pancreatic necrosis (PNec), and 7% died. OF (54% vs 33%, P = 0.03), PNec (62% vs 31%, P = 0.006), intensive care unit admission (58% vs 36%, P = 0.03), and length of stay (LOS) (20 vs 8 days, P = 0.007) were greater in alcoholic when compared to other etiologies. Spanish cohort (n = 366): Similar differences in outcomes were also found with between alcoholic and nonalcoholic etiologies: OF (24% vs 8%, P = 0.001), PNec (38% vs 14%, P < 0.001), intensive care unit admission (20% vs 3%, P < 0.001), and LOS (17 vs 11 days, P = 0.04). Multivariable analysis confirmed alcoholic etiology to be independently associated with OF and PNec in both cohorts. Conclusions Alcoholic etiology is independently associated with OF and PNec in patients with sentinel AP and is important when evaluating risk for severe disease in AP.

Fatty Pancreas: Should We Be Concerned?

Abstract The metabolic consequences of visceral fat deposition are well known, and the presence of intrapancreatic fat (IPF) has been recognized for decades. However, our knowledge about the distribution of fat in the pancreas and its clinical implications is in a nascent stage. Various terms have been proposed to describe IPF; for the purpose of this narrative review, we chose the general term fatty pancreas. Herein, we describe the radiologic, endoscopic, and histopathologic aspects of diagnosing fatty pancreas and provide an overview of the diseases associated with this condition. Our purpose is to highlight diagnostic challenges and identify specific clinical questions that would benefit from further study. As evident in this review, IPF is associated with various metabolic diseases, pancreatitis, pancreatic cancer, and precancer—yet establishing causality needs careful, further study.

Trafficking of Cathepsin B Containing Organelles to the Autophagosome in an ARF-1 Dependent Manner is Required for Caerulein Induced Trypsinogen Activation

Background: While cathepsin B mediated trypsin generation occurs in acute pancreatitis, the regulation of organellar dynamics involved in this process is unclear. Previous studies show co-localization of cathepsin B and zymogens to occur in vacuoles. Additionally, caerulein (CER) causes autophagy in rodent pancreatitis. We therefore studied the organellar location and regulation of cathepsin B trafficking in response to caerulein and its effect on autophagy and pancreatitis. Methods: In Vitro pancreatic acini were treated with CER with or without 50μM brefeldin A (BFA), or In Vivo CER pancreatitis was induced using standard methods in rats (6 hour) and mice (10 hour) given BFA (25 mg/kg) or vehicle (0.1ml 50% DMSO) intraperitoneally (IP). Autophagic markers (LC3), autophagosome formation (electron microscopy; EM), ARF-1 activation and pancreatitis severity parameters were studied. Trypsin generation (arbitrary units/μg DNA), Golgi morphology (P115 immuno-staining), Cathepsin B amounts (western blotting, WB), localization (sub-cellular fractionation), amylase secretion were also acessed. Results: Under basal conditions Cathepsin B localized to the lysozomal, cis-golgi and ER fractions but not to the zymogen granule (ZG) or syntaxin 6 rich compartments. Within 30 minutes, 0.1nM and 100nM CER completely depleted pancreatic acini of mature Cathepsin B (33KD), and partially depleted its 24KD, 27KD double chain forms but not ZG contents (lipase, chymotrypsin) suggesting distinct Cathepsin B and ZG trafficking mechanisms. BFA prevented cathepsin B depletion, inhibited ARF-1 activation, reduced 100nM CER induced cathepsin B trafficking to the 1300g pellet (ratio 1.08 with 100nM CER vs. 0.52 with 100nM CER+BFA, p<0.05), trypsin generation In Vitro (2.27 vs.1.03 fold basal) and In Vivo (8.05 vs. 3.78 fold with CER+ vehicle and CER+BFA respectively, p<0.03). BFA also collapsed the Golgi, without affecting CER induced F-actin reorganization or amylase secretion. In Vivo, BFA did not prevent autophagy on WB (LC3-II formation) or on EM. However CER induced autophagosomes contained both ZG and non ZG organelles (78%), vs. 21% with CER+BFA. CER+BFA resulted in 48% autophagosomes containing only ZGs vs 12% with CER, and 31% being empty vs 10% in CER. BFA reduced severity parameters of pancreatitis in both mice (amylase12.9 vs.5.2 fold basal, pancreatic edema 83.8% vs. 76% and pancreatic necrosis 15% vs. 1.4% with CER+ vehicle and CER+BFA respectively, p<0.01) and rats (amylase 8.0 fold vs. 3.8 fold, edema 82.6% vs. 75.6%, P<0.05). Conclusions: Caerulein mediated trafficking of organelles containing Cathepsin B to the autophagosome, in contrast to that of ZGs is dependent on ARF-1 and is required for trypsin generation. Inhibition of ARF-1 prevents this trafficking and reduces severity of pancreatitis without inhibiting autophagy.

Primary (Obesity Associated) Intrapancreatic Fat is Quantitatively, Morphologically, Pathogenically and Clinically Distinct From Secondary (Chronic Pancreatitis Associated) Intrapancreatic Fat

matched controls (GSP) were selected for each HIP case. Hospital course was then compared between the two groups, based on local/systemic complications of acute pancreatitis, management and length of stay. Statistics: Fischers exact t-test was used and a two sided p-value was calculated; p-value of less than 0.05 was considered statistically significant. Results: There were 292 patients admitted during the study period. Etiologies were GSP (41.8%), alcoholic pancreatitis (23.3%), others (autoimmune, drugs, ERCP, idiopathic) (30.1%) and HIP (4.8%). There were 14 HIP cases (64% men)and 42 GSP controls (60% men). Mean age was 43.8 (range 24-64) and 44 (range 17-71) for cases and controls, respectively. Mean admission lipase levels were lower in HIP(638 mg/dl) than in GSP(2399 mg/dl). BISAP score on admission in all HIP cases was less than 2. As shown in the table, there was a significant difference between the two groups in the following variables: 2 or more abdominal CT scans, systemic complications (such as pleural effusions, ARDS, renal failure), transfer to the ICU (for renal failure,candidemia and respiratory distress) and use of TPN. There were no deaths in either group. Conclusions: Based on our data, HIP appears to follow a more aggressive course with greater resource utilization than GSP. Furthermore, a low BISAP score in HIP may not be predictive of in-hospital morbidity. These findings need to be confirmed in a prospective study with a larger sample size. Statistical comparison of various variables during the hospital course between cases (HIP) and controls (GSP)

Serine proteases regulate exocytosis and F-actin distribution in rat pancreatic acinar cells

Background: The polarized pancreatic acinar cell secretes digestive enzymes from its apical pole in response to various secretagogues. However not much is known about the steps that occur in this process subsequent to the increase of cytosolic calcium. We have recently shown that a serine protease activity is essential for the exocytosis of digestive enzymes from pancreatic acinar cells in response to calcium-mediated secretagogues. However the mechanism by which this protease regulates exocytosis is not known. In the present study we have evaluated the effect of serine protease inhibition on the acinar cell cytoskeleton. Methods: Acini were prepared by collagenase digestion and were stimulated by various secretagogues in the presence or absence of cell permeant serine protease inhibitors 4-(2aminoethyl)-benzenesulfonyl flouride (AEBSF) and TPCK. F-Actin distribution was studied after staining with rhodamine phalloidin and examination under the confocal microscope. Results: Both cell permeant serine protease inhibitors (AEBSF and TPCK) blocked exocytosis in response to secretagogues that use calcium as a second messenger (e.g. caerulein, carbamylcholine and bombesin). The non-cell permeant inhibitor SBTI did not affect exocytosis. Incubation of the acini with the cell permeant serine protease inhibitors resulted in a dramatic redistribution of the F-actin cytoskeleton. Under basal conditions F-actin was primarily localized at the apical pole of the acinar cell. After exposure to the cell permeant serine protease inhibitors, F-actin was redistributed to the basolateral region of the cell. This redistribution was blocked by oligomycin, showing that the phenomenon is energy dependent. Similar redistribution of F-actin from the apical to the basolateral region was also observed when acini were incubated with a supramaximally stimulating concentration of caerulein which is known to inhibit secretion via its low affinity receptor. Conclusion: These results suggest that a serine protease activity is essential for maintaining the normal apical F-actin distribution and hence apical exocytosis in the rat pancreatic acinar cell. Blockage of this activity redistributes F-actin from the apical to the basolateral region and blocks exocytosis in response to agents acting via calcium.