Protective Effects of Necrostatin-1 in Acute Pancreatitis: Partial Involvement of Receptor Interacting Protein Kinase 1.

Acute pancreatitis (AP) is a severe and potentially fatal disease caused predominantly by alcohol excess and gallstones, which lacks a specific therapy. The role of Receptor-Interacting Protein Kinase 1 (RIPK1), a key component of programmed necrosis (Necroptosis), is unclear in AP. We assessed the effects of RIPK1 inhibitor Necrostatin-1 (Nec-1) and RIPK1 modification (RIPK1K45A: kinase dead) in bile acid (TLCS-AP), alcoholic (FAEE-AP) and caerulein hyperstimulation (CER-AP) mouse models. Involvement of collateral Nec-1 target indoleamine 2,3-dioxygenase (IDO) was probed with the inhibitor Epacadostat (EPA). Effects of Nec-1 and RIPK1K45A were also compared on pancreatic acinar cell (PAC) fate in vitro and underlying mechanisms explored. Nec-1 markedly ameliorated histological and biochemical changes in all models. However, these were only partially reduced or unchanged in RIPK1K45A mice. Inhibition of IDO with EPA was protective in TLCS-AP. Both Nec-1 and RIPK1K45A modification inhibited TLCS- and FAEE-induced PAC necrosis in vitro. Nec-1 did not affect TLCS-induced Ca2+ entry in PACs, however, it inhibited an associated ROS elevation. The results demonstrate protective actions of Nec-1 in multiple models. However, RIPK1-dependent necroptosis only partially contributed to beneficial effects, and actions on targets such as IDO are likely to be important.

Pancreatic Acinar Cell Preparation for Oxygen Consumption and Lactate Production Analysis.

Mitochondrial dysfunction is a principal feature of acute pancreatitis (AP) although the underlying mechanisms are still unclear. AP precipitants induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP) in pancreatic acinar cells (PACs), leading to ATP depletion and necrosis. Evaluations of mitochondrial bioenergetics have mainly been performed in isolated PACs using confocal microscopy, with assessment of mitochondrial membrane potential, NADH/FAD+ and ATP levels, coupled with patch-clamp electrophysiology. These studies are technically demanding and time-consuming. Application of Seahorse flux analysis now allows detailed investigations of bioenergetics changes to be performed in cell populations using a multi-well plate-reader format; rates of oxygen consumption (OCR) and extracellular acidification (ECAR) provide important information about cellular respiration and glycolysis, respectively. Parameters such as maximal respiration, ATP-linked capacity and proton leak can be derived from application of a respiratory function "stress" test that involves pharmacological manipulation of the electron transport chain. The use of Seahorse Flux analysis therefore provides a quick, and convenient means to measure detailed cellular bioenergetics and allows results to be coupled with other plate-reader based assays, providing a fuller understanding of the pathophysiological consequences of mitochondrial bioenergetics alterations.

Altered Bioenergetics of Blood Cell Sub-Populations in Acute Pancreatitis Patients.

Acute pancreatitis (AP) is a debilitating, sometimes fatal disease, marked by local injury and systemic inflammation. Mitochondrial dysfunction is a central feature of pancreatic damage in AP, however, its involvement in circulating blood cell subtypes is unknown. This study compared mitochondrial bioenergetics in circulating leukocytes from AP patients and healthy volunteers: 15 patients with mild to severe AP were compared to 10 healthy controls. Monocytes, lymphocytes and neutrophils were isolated using magnetic activated cell sorting and mitochondrial bioenergetics profiles of the cell populations determined using a Seahorse XF24 flux analyser. Rates of oxygen consumption (OCR) and extracellular acidification (ECAR) under conditions of electron transport chain (ETC) inhibition ("stress" test) informed respiratory and glycolytic parameters, respectively. Phorbol ester stimulation was used to trigger the oxidative burst. Basal OCR in all blood cell subtypes was similar in AP patients and controls. However, maximal respiration and spare respiratory capacity of AP patient lymphocytes were decreased, indicating impairment of functional capacity. A diminished oxidative burst occurred in neutrophils from AP patients, compared to controls, whereas this was enhanced in both monocytes and lymphocytes. The data demonstrate important early alterations of bioenergetics in blood cell sub-populations from AP patients, which imply functional alterations linked to clinical disease progression.

Mitochondrial Targeting of Antioxidants Alters Pancreatic Acinar Cell Bioenergetics and Determines Cell Fate.

Mitochondrial dysfunction is a core feature of acute pancreatitis, a severe disease in which oxidative stress is elevated. Mitochondrial targeting of antioxidants is a potential therapeutic strategy for this and other diseases, although thus far mixed results have been reported. We investigated the effects of mitochondrial targeting with the antioxidant MitoQ on pancreatic acinar cell bioenergetics, adenosine triphosphate (ATP) production and cell fate, in comparison with the non-antioxidant control decyltriphenylphosphonium bromide (DecylTPP) and general antioxidant N-acetylcysteine (NAC). MitoQ (µM range) and NAC (mM range) caused sustained elevations of basal respiration and the inhibition of spare respiratory capacity, which was attributable to an antioxidant action since these effects were minimal with DecylTPP. Although MitoQ but not DecylTPP decreased cellular NADH levels, mitochondrial ATP turnover capacity and cellular ATP concentrations were markedly reduced by both MitoQ and DecylTPP, indicating a non-specific effect of mitochondrial targeting. All three compounds were associated with a compensatory elevation of glycolysis and concentration-dependent increases in acinar cell apoptosis and necrosis. These data suggest that reactive oxygen species (ROS) contribute a significant negative feedback control of basal cellular metabolism. Mitochondrial targeting using positively charged molecules that insert into the inner mitochondrial member appears to be deleterious in pancreatic acinar cells, as does an antioxidant strategy for the treatment of acute pancreatitis.

Oxidative stress alters mitochondrial bioenergetics and modifies pancreatic cell death independently of cyclophilin D, resulting in an apoptosis-to-necrosis shift.

Mitochondrial dysfunction lies at the core of acute pancreatitis (AP). Diverse AP stimuli induce Ca2+-dependent formation of the mitochondrial permeability transition pore (MPTP), a solute channel modulated by cyclophilin D (CypD), the formation of which causes ATP depletion and necrosis. Oxidative stress reportedly triggers MPTP formation and is elevated in clinical AP, but how reactive oxygen species influence cell death is unclear. Here, we assessed potential MPTP involvement in oxidant-induced effects on pancreatic acinar cell bioenergetics and fate. H2O2 application promoted acinar cell apoptosis at low concentrations (1-10 µm), whereas higher levels (0.5-1 mm) elicited rapid necrosis. H2O2 also decreased the mitochondrial NADH/FAD+ redox ratio and ΔΨm in a concentration-dependent manner (10 µm to 1 mm H2O2), with maximal effects at 500 µm H2O2 H2O2 decreased the basal O2 consumption rate of acinar cells, with no alteration of ATP turnover at <50 µm H2O2 However, higher H2O2 levels (≥50 µm) diminished spare respiratory capacity and ATP turnover, and bioenergetic collapse, ATP depletion, and cell death ensued. Menadione exerted detrimental bioenergetic effects similar to those of H2O2, which were inhibited by the antioxidant N-acetylcysteine. Oxidant-induced bioenergetic changes, loss of ΔΨm, and cell death were not ameliorated by genetic deletion of CypD or by its acute inhibition with cyclosporine A. These results indicate that oxidative stress alters mitochondrial bioenergetics and modifies pancreatic acinar cell death. A shift from apoptosis to necrosis appears to be associated with decreased mitochondrial spare respiratory capacity and ATP production, effects that are independent of CypD-sensitive MPTP formation.

Physiological intestinal oxygen modulates the Caco-2 cell model and increases sensitivity to the phytocannabinoid cannabidiol.

The Caco-2 cell model is widely used as a model of colon cancer and small intestinal epithelium but, like most cell models, is cultured in atmospheric oxygen conditions (∼21%). This does not reflect the physiological oxygen range found in the colon. In this study, we investigated the effect of adapting the Caco-2 cell line to routine culturing in a physiological oxygen (5%) environment. Under these conditions, cells maintain a number of key characteristics of the Caco-2 model, such as increased formation of tight junctions and alkaline phosphatase expression over the differentiation period and maintenance of barrier function. However, these cells exhibit differential oxidative metabolism, proliferate less and become larger during differentiation. In addition, these cells were more sensitive to cannabidiol-induced antiproliferative actions through changes in cellular energetics: from a drop of oxygen consumption rate and loss of mitochondrial membrane integrity in cells treated under atmospheric conditions to an increase in reactive oxygen species in intact mitochondria in cells treated under low-oxygen conditions. Inclusion of an additional physiological parameter, sodium butyrate, into the medium revealed a cannabidiol-induced proliferative response at low doses. These effects could impact on its development as an anticancer therapeutic, but overall, the data supports the principle that culturing cells in microenvironments that more closely mimic the in vivo conditions is important for drug screening and mechanism of action studies.

Fatty acid ethyl ester synthase inhibition ameliorates ethanol-induced Ca2+-dependent mitochondrial dysfunction and acute pancreatitis.

OBJECTIVE: Non-oxidative metabolism of ethanol (NOME) produces fatty acid ethyl esters (FAEEs) via carboxylester lipase (CEL) and other enzyme action implicated in mitochondrial injury and acute pancreatitis (AP). This study investigated the relative importance of oxidative and non-oxidative pathways in mitochondrial dysfunction, pancreatic damage and development of alcoholic AP, and whether deleterious effects of NOME are preventable. DESIGN: Intracellular calcium ([Ca(2+)](C)), NAD(P)H, mitochondrial membrane potential and activation of apoptotic and necrotic cell death pathways were examined in isolated pancreatic acinar cells in response to ethanol and/or palmitoleic acid (POA) in the presence or absence of 4-methylpyrazole (4-MP) to inhibit oxidative metabolism. A novel in vivo model of alcoholic AP induced by intraperitoneal administration of ethanol and POA was developed to assess the effects of manipulating alcohol metabolism. RESULTS: Inhibition of OME with 4-MP converted predominantly transient [Ca(2+)](C) rises induced by low ethanol/POA combination to sustained elevations, with concurrent mitochondrial depolarisation, fall of NAD(P)H and cellular necrosis in vitro. All effects were prevented by 3-benzyl-6-chloro-2-pyrone (3-BCP), a CEL inhibitor. 3-BCP also significantly inhibited rises of pancreatic FAEE in vivo and ameliorated acute pancreatic damage and inflammation induced by administration of ethanol and POA to mice. CONCLUSIONS: A combination of low ethanol and fatty acid that did not exert deleterious effects per se became toxic when oxidative metabolism was inhibited. The in vitro and in vivo damage was markedly inhibited by blockade of CEL, indicating the potential for development of specific therapy for treatment of alcoholic AP via inhibition of FAEE generation.