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.

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)-1ß and keratinocyte chemoattractant/growth-regulated oncogene, triolein alone or with the lipase inhibitor orlistat. RESULTS: NCs had higher fatty acids, IL-8 and IL-1ß 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-1ß 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.

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.

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.

Src Dependent Pancreatic Acinar Injury Can Be Initiated Independent of an Increase in Cytosolic Calcium.

Several deleterious intra-acinar phenomena are simultaneously triggered on initiating acute pancreatitis. These culminate in acinar injury or inflammatory mediator generation in vitro and parenchymal damage in vivo. Supraphysiologic caerulein is one such initiator which simultaneously activates numerous signaling pathways including non-receptor tyrosine kinases such as of the Src family. It also causes a sustained increase in cytosolic calcium- a player thought to be crucial in regulating deleterious phenomena. We have shown Src to be involved in caerulein induced actin remodeling, and caerulein induced changes in the Golgi and post-Golgi trafficking to be involved in trypsinogen activation, which initiates acinar cell injury. However, it remains unclear whether an increase in cytosolic calcium is necessary to initiate acinar injury or if injury can be initiated at basal cytosolic calcium levels by an alternate pathway. To study the interplay between tyrosine kinase signaling and calcium, we treated mouse pancreatic acinar cells with the tyrosine phosphatase inhibitor pervanadate. We studied the effect of the clinically used Src inhibitor Dasatinib (BMS-354825) on pervanadate or caerulein induced changes in Src activation, trypsinogen activation, cell injury, upstream cytosolic calcium, actin and Golgi morphology. Pervanadate, like supraphysiologic caerulein, induced Src activation, redistribution of the F-actin from its normal location in the sub-apical area to the basolateral areas, and caused antegrade fragmentation of the Golgi. These changes, like those induced by supraphysiologic caerulein, were associated with trypsinogen activation and acinar injury, all of which were prevented by Dasatinib. Interestingly, however, pervanadate did not cause an increase in cytosolic calcium, and the caerulein induced increase in cytosolic calcium was not affected by Dasatinib. These findings suggest that intra-acinar deleterious phenomena may be initiated independent of an increase in cytosolic calcium. Other players resulting in acinar injury along with the Src family of tyrosine kinases remain to be explored.

Lipotoxicity causes multisystem organ failure and exacerbates acute pancreatitis in obesity.

Obesity increases the risk of adverse outcomes during acute critical illnesses such as burns, severe trauma, and acute pancreatitis. Although individuals with more body fat and higher serum cytokines and lipase are more likely to experience problems, the roles that these characteristics play are not clear. We used severe acute pancreatitis as a representative disease to investigate the effects of obesity on local organ function and systemic processes. In obese humans, we found that an increase in the volume of intrapancreatic adipocytes was associated with more extensive pancreatic necrosis during acute pancreatitis and that acute pancreatitis was associated with multisystem organ failure in obese individuals. In vitro studies of pancreatic acinar cells showed that unsaturated fatty acids were proinflammatory, releasing intracellular calcium, inhibiting mitochondrial complexes I and V, and causing necrosis. Saturated fatty acids had no such effects. Inhibition of lipolysis in obese (ob/ob) mice with induced pancreatitis prevented a rise in serum unsaturated fatty acids and prevented renal injury, lung injury, systemic inflammation, hypocalcemia, reduced pancreatic necrosis, and mortality. Thus, therapeutic approaches that target unsaturated fatty acid-mediated lipotoxicity may reduce adverse outcomes in obese patients with critical illnesses such as severe acute pancreatitis.