Krutika Patel

Peripancreatic fat necrosis worsens acute pancreatitis independent of pancreatic necrosis via unsaturated fatty acids increased in human pancreatic necrosis collections

Background and aims Peripancreatic fat necrosis occurs frequently in necrotising pancreatitis. Distinguishing markers from mediators of severe acute pancreatitis (SAP) is important since targeting mediators may improve outcomes. We evaluated potential agents in human pancreatic necrotic collections (NCs), pseudocysts (PCs) and pancreatic cystic neoplasms and used pancreatic acini, peripheral blood mononuclear cells (PBMC) and an acute pancreatitis (AP) model to determine SAP mediators. Methods We measured acinar and PBMC injury induced by agents increased in NCs and PCs. Outcomes of caerulein pancreatitis were studied in lean rats coadministered interleukin (IL)-1β and keratinocyte chemoattractant/growth-regulated oncogene, triolein alone or with the lipase inhibitor orlistat. Results NCs had higher fatty acids, IL-8 and IL-1β versus other fluids. Lipolysis of unsaturated triglyceride and resulting unsaturated fatty acids (UFA) oleic and linoleic acids induced necro-apoptosis at less than half the concentration in NCs but other agents did not do so at more than two times these concentrations. Cytokine coadministration resulted in higher pancreatic and lung inflammation than caerulein alone, but only triolein coadministration caused peripancreatic fat stranding, higher cytokines, UFAs, multisystem organ failure (MSOF) and mortality in 97% animals, which were prevented by orlistat. Conclusions UFAs, IL-1β and IL-8 are elevated in NCs. However, UFAs generated via peripancreatic fat lipolysis causes worse inflammation and MSOF, converting mild AP to SAP.

Lipolysis of Visceral Adipocyte Triglyceride by Pancreatic Lipases Converts Mild Acute Pancreatitis to Severe Pancreatitis Independent of Necrosis and Inflammation

Visceral fat necrosis has been associated with severe acute pancreatitis (SAP) for over 100 years; however, its pathogenesis and role in SAP outcomes are poorly understood. Based on recent work suggesting that pancreatic fat lipolysis plays an important role in SAP, we evaluated the role of pancreatic lipases in SAP-associated visceral fat necrosis, the inflammatory response, local injury, and outcomes of acute pancreatitis (AP). For this, cerulein pancreatitis was induced in lean and obese mice, alone or with the lipase inhibitor orlistat and parameters of AP induction (serum amylase and lipase), fat necrosis, pancreatic necrosis, and multisystem organ failure, and inflammatory response were assessed. Pancreatic lipases were measured in fat necrosis and were overexpressed in 3T3-L1 cells. We noted obesity to convert mild cerulein AP to SAP with greater cytokines, unsaturated fatty acids (UFAs), and multisystem organ failure, and 100% mortality without affecting AP induction or pancreatic necrosis. Increased pancreatic lipase amounts and activity were noted in the extensive visceral fat necrosis of dying obese mice. Lipase inhibition reduced fat necrosis, UFAs, organ failure, and mortality but not the parameters of AP induction. Pancreatic lipase expression increased lipolysis in 3T3-L1 cells. We conclude that UFAs generated via lipolysis of visceral fat by pancreatic lipases convert mild AP to SAP independent of pancreatic necrosis and the inflammatory response.

Acute Lipotoxicity Regulates Severity of Biliary Acute Pancreatitis without Affecting Its Initiation

Obese patients have worse outcomes during acute pancreatitis (AP). Previous animal models of AP have found worse outcomes in obese rodents who may have a baseline proinflammatory state. Our aim was to study the role of acute lipolytic generation of fatty acids on local severity and systemic complications of AP. Human postpancreatitis necrotic collections were analyzed for unsaturated fatty acids (UFAs) and saturated fatty acids. A model of biliary AP was designed to replicate the human variables by intraductal injection of the triglyceride glyceryl trilinoleate alone or with the chemically distinct lipase inhibitors orlistat or cetilistat. Parameters of AP etiology and outcomes of local and systemic severity were measured. Patients with postpancreatitis necrotic collections were obese, and 13 of 15 had biliary AP. Postpancreatitis necrotic collections were enriched in UFAs. Intraductal glyceryl trilinoleate with or without the lipase inhibitors resulted in oil red O-positive areas, resembling intrapancreatic fat. Both lipase inhibitors reduced the glyceryl trilinoleate-induced increase in serum lipase, UFAs, pancreatic necrosis, serum inflammatory markers, systemic injury, and mortality but not serum alanine aminotransferase, bilirubin, or amylase. We conclude that UFAs are enriched in human necrotic collections and acute UFA generation via lipolysis worsens pancreatic necrosis, systemic inflammation, and injury associated with severe AP. Inhibition of lipolysis reduces UFA generation and improves these outcomes of AP without interfering with its induction.

Fatty Acid Ethyl Esters Are Less Toxic Than Their Parent Fatty Acids Generated during Acute Pancreatitis

Although ethanol causes acute pancreatitis (AP) and lipolytic fatty acid (FA) generation worsens AP, the contribution of ethanol metabolites of FAs, ie, FA ethyl esters (FAEEs), to AP outcomes is unclear. Previously, pancreata of dying alcoholics and pancreatic necrosis in severe AP, respectively, showed high FAEEs and FAs, with oleic acid (OA) and its ethyl esters being the most abundant. We thus compared the toxicities of FAEEs and their parent FAs in severe AP. Pancreatic acini and peripheral blood mononuclear cells were exposed to FAs or FAEEs in vitro. The triglyceride of OA (i.e., glyceryl tri-oleate) or OAEE was injected into the pancreatic ducts of rats, and local and systemic severities were studied. Unsaturated FAs at equimolar concentrations to FAEEs induced a larger increase in cytosolic calcium, mitochondrial depolarization, and necro-apoptotic cell death. Glyceryl tri-oleate but not OAEE resulted in 70% mortality with increased serum OA, a severe inflammatory response, worse pancreatic necrosis, and multisystem organ failure. Our data show that FAs are more likely to worsen AP than FAEEs. Our observations correlate well with the high pancreatic FAEE concentrations in alcoholics without pancreatitis and high FA concentrations in pancreatic necrosis. Thus, conversion of FAs to FAEE may ameliorate AP in alcoholics.

Potential Influence of Intravenous Lipids on the Outcomes of Acute Pancreatitis

Parenteral nutrition (PN) has been associated with a higher rate of adverse outcomes compared with enteral feeding in patients with acute pancreatitis (AP). However, PN may be necessary when feeding via the enteral route is poorly tolerated or impossible, and PN is recommended as a second-line nutrition therapy in AP. Intravenous (IV) lipids are commonly used as a part of PN in patients with AP. While the adverse outcomes related to the use of PN in AP have commonly been attributed to infectious complications, data suggest that the unsaturated fatty acids in the triglycerides used in IV lipids may contribute to the development of organ failure. We discuss the clinical and experimental data on this issue and the alternative lipid emulsions that are being studied.

Hypothermia slows sequential and parallel steps initiated during caerulein pancreatitis.

BACKGROUND AND OBJECTIVES Multiple deleterious signaling cascades are simultaneously activated in acute pancreatitis (AP), which may limit the success of pharmacologic approaches targeting a single step. We explored whether cooling acinar cells slows distinct steps initiated from a stimulus causing pancreatitis simultaneously, and the temperature range over which inhibition of such deleterious signaling occurs. METHODS Caerulein (100 nM) induced trypsinogen activation (TGA), CXCL1, CXCL2 mRNA levels, cell injury were studied at 37 °C, 34 °C, 31 °C, 29 °C and 25 °C in acinar cells. Trypsin, cathepsin B activities and cathepsin B mediated TGA were studied at 37 °C, 23 °C and 4 °C. RESULTS There was >80% reduction in TGA, CXCL1 and CXCL2 mRNA levels at 29 °C, and in cell injury at 34 °C, compared to those at 37 °C. Trypsin activity, cathepsin B activity and cathepsin B mediated TGA at 23 °C were respectively, 53%, 64% and 26% of that at 37 °C. Acinar cooling to 31 °C reduced LDH leakage even when cooling was initiated an hour after caerulein stimulation at 37 °C. CONCLUSIONS Hypothermia synergistically and simultaneously slows parallel and distinct signaling steps initiated by caerulein, thereby reducing TGA, upregulation of inflammatory mediators and acinar injury.

Multimodal Transgastric Local Pancreatic Hypothermia Reduces Severity of Acute Pancreatitis in Rats and Increases Survival

BACKGROUND & AIMS Acute pancreatitis (AP) of different etiologies is associated with the activation of different signaling pathways in pancreatic cells, posing challenges to the development of targeted therapies. We investigated whether local pancreatic hypothermia, without systemic hypothermia, could lessen the severity of AP induced by different methods in rats. METHODS A urethane balloon with 2 polyurethane tubes was placed inside the stomach of rats. AP was induced in Wistar rats by the administration of cerulein or glyceryl tri-linoleate (GTL). Then, cold water was infused into the balloon to cool the pancreas. Pancreatic temperatures were selected based on those found to decrease acinar cell injury. An un-perfused balloon was used as a control. Pancreatic and rectal temperatures were monitored, and an infrared lamp or heating pad was used to avoid generalized hypothermia. We collected blood, pancreas, kidney, and lung tissues and analyzed them by histology, immunofluorescence, immunoblot, cytokine and chemokine magnetic bead, and DNA damage assays. The effect of hypothermia on signaling pathways initiated by cerulein and GTL was studied in acinar cells. RESULTS Rats with pancreatic cooling developed less severe GTL-induced AP compared with rats that received the control balloon. In acinar cells, cooling decreased the lipolysis induced by GTL, increased the micellar form of its fatty acid, lowered the increase in cytosolic calcium, prevented the loss of mitochondrial membrane potential (by 70%-80%), and resulted in a 40%-50% decrease in the uptake of a fatty acid tracer. In rats with AP, cooling decreased pancreatic necrosis by 48%, decreased serum levels of cytokines and markers of cell damage, and decreased markers of lung and renal damage. Pancreatic cooling increased the proportions of rats surviving 6 hours after induction of AP (to 90%, from <10% of rats that received the control balloon). In rats with cerulein-induced AP, pancreatic cooling decreased pancreatic markers of apoptosis and inflammation. CONCLUSIONS In rats with AP, transgastric local pancreatic hypothermia decreases pancreatic necrosis, apoptosis, inflammation, and markers of pancreatitis severity and increases survival.

Characterization and predictive value of near infrared 2-deoxyglucose optical imaging in severe acute pancreatitis

Background Studying the uptake of 2-deoxy glucose (2-DG) analogs such as 2-Deoxy-2-[18F] fluoroglucose (FDG) is a common approach to identify and monitor malignancies and more recently chronic inflammation. While pancreatitis is a common cause for false positive results in human studies on pancreatic cancer using FDG, the relevance of these findings to acute pancreatitis (AP) is unknown. FDG has a short half-life. Thus, with an aim to accurately characterize the metabolic demand of the pancreas during AP in real-time, we studied the uptake of the non-radioactive, near infrared fluorescence labelled 2-deoxyglucose analog, IRDye® 800CW 2-DG probe (NIR 2-DG; Li-Cor) during mild and severe biliary AP. Methods Wistar rats (300 g; 8–12/group) were administered NIR 2-DG (10 nM; I.V.). Mild and severe biliary AP were respectively induced by biliopancreatic duct ligation (DL) alone or along with infusing glyceryl trilinoleate (GTL; 50 μL/100 g) within 10 minutes of giving NIR 2-DG. Controls (CON) only received NIR 2-DG. Imaging was done every 5–10 minutes over 3 hrs. Average Radiant Efficiency [p/s/cm²/sr]/[μW/cm²] was measured over the pancreas using the IVIS 200 in-vivo imaging system (PerkinElmer) using the Living Image® software and verified in ex vivo pancreata. Blood amylase, lipase and pancreatic edema, necrosis were measured over the course of AP. Results NIR 2-DG uptake over the first hour was not influenced by AP induction. However, while the signal declined in controls and rats with mild AP, there was significantly higher retention of NIR 2-DG in the pancreas after 1 hour in those with GTL pancreatitis. The increase was > 3 fold over controls in the GTL group and was verified to be in the pancreas ex vivo. In vitro, pancreatic acini exposed to GTL had a similar increase in NIR 2-DG uptake which was followed by progressively worse acinar necrosis. Greater retention of NIR 2-DG in vivo was associated with worse pancreatic necrosis, reduced ATP concentrations and mortality, which were not predicted by the blood parameters. Conclusion In-vivo fluorescent imaging of a non-radioactive near infrared 2-DG optical probe can predict the AP severity early during the disease.