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Pyruvate and Glutamine Define the Effects of Cholecystokinin and Ethanol on Mitochondrial Oxidation, Necrosis, and Morphology of Rat Pancreatic Acini
Abstract
Objectives:
The objective of this study was to test whether pyruvate and glutamine affect the ethanol and cholecystokinin (CCK) effects on the mitochondrial function, viability, and morphology of rat pancreatic acini.
Methods:
Respiration was measured with Clark oxygen electrode. Mitochondrial membrane potential, reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H), cell morphology, and viability were studied with fluorescence microscopy.
Results:
In vitro, CCK (0.1 nM) caused pyruvate-dependent stimulation of basal and uncoupled respiration, and the effects were abolished by ethanol (20 mM). The combination of ethanol with CCK (2 hours) caused necrosis of approximately 40% acinar cells in medium with glucose, but not with pyruvate and/or glutamine. Cholecystokinin (10 nM) or ethanol with 0.1 nM CCK caused plasma membrane blebbing not related to apoptosis only when both glutamine and pyruvate were present. Glutamine, but not pyruvate, decreased NAD(P)H level and prevented the effects of ethanol with CCK on mitochondrial membrane potential and NAD(P)H, but, in combination with CCK and ethanol, decreased the uncoupled respiration. In vivo, the combination of ethanol (4 g/kg) and CCK (20 pmol/kg) suppressed basal and uncoupled respiration and caused acinar cell blebbing, but not necrosis.
Conclusions:
The lack of sufficient substrate supply in vitro makes pancreatic acinar cells susceptible to necrosis caused by ethanol and CCK in clinically relevant concentrations.
... Nevertheless, these outcomes were not verified in vivo, and it is most likely that ethanol requires additional factors to cause pancreatitis [12,15,17]. In our previous study, we found that while a combination of ethanol and cholecystokinin administration to rats caused a significant decrease of both basal and uncoupled respiration rate of isolated pancreatic acini, ethanol alone did not affect the uncoupled respiration and its effect on basal oxygen consumption was less convincing [16] due to a complex experimental design. ...
... These results suggest that acute alcohol administration does not damage mitochondria in pancreatic acinar cells, which might lead to acute pancreatitis [18]. Only a combination of ethanol with an additional factors impairs pancreatic acinar cells in animal models [9,12,16]. It is known that ATP levels decrease in pancreatic acinar cells following various pancreatic injuries [5], but the mechanism has not been fully studied. ...
... Data from in vitro experiments show that 10-100 μM FAEE induces a rise in intracellular [Ca²⁺] [4,13]. Our previous experiments have shown that pyruvate supplementation improves the survival of pancreatic acini when they are exposed to ethanol and CCK [16]. We can assume that alcohol consumption to leads the formation of FAEE, which triggers Ca 2+ release and the activation of the pyruvate dehydrogenase in rats. ...
The exocrine function of pancreatic acini cells is a highly energy-intensive process. A cell always needs to maintain a stable level of ATP balancing between states of activation and rest. Low-energy is one of the possible mechanisms contributing to the development of pancreatic diseases. The most commonly encountered disease of the pancreas is acute pancreatitis. It is known that excessive alcohol consumption causes the development of pancreatitis. The pathogenesis of this disease is linked to the cellular loss of energy, but the mechanism of alcohol’s effect on the mitochondria in pancreatic acini is unclear. This study’s main aim is to assess the impact of acute alcohol administration on the mitochondrial function of rat pancreatic acini. Wistar rats were administered ethanol (6 g/kg body weight) by oral gavage for 3 h before the experiment. A suspension of isolated pancreatic acini was obtained following collagenase digestion. Respiration of isolated pancreatic acini was studied with a Clark electrode. The maximal respiration rate was studied at different concentrations of protonophore FCCP (0.5–2 μM) in solutions containing glucose combined with oxidative substrates (pyruvate and glutamine, monomethyl-succinate or dimethyl-α-ketoglutarate). Dehydrogenase activity was measured by colorimetric method. Ethanol administration caused a significant increase in the activity of pyruvate dehydrogenase. It was confirmed that FCCP induced an increase in the respiration rate of pancreatic acinar cells in each experimental group. The addition of 1.5 μM FCCP reduced the respiration rate of pancreatic acini during the oxidation of glucose and monomethyl succinate or dimethyl-α-ketoglutarate, but not during the oxidation of glucose, pyruvate and glutamine substrates. The administration of ethanol had no impact on the basal or FCCP-uncoupled respiration of isolated pancreatic acini. The observed data are consistent with the findings of other researchers. However, alcohol exposure is not sufficient to cause mitochondrial damage in pancreatic acinar cells. In conclusion, acute ethanol administration does not cause mitochondrial dysfunction in the pancreas of rats but causes an increase in pyruvate dehydrogenase activity.
... The studied amino acids were added at 20 mM. Pancreatic acinar cell necrosis was studied with ethidium bromide and Hoechst 33258 staining as described earlier (Manko et al. 2021). Cell blebbing counting was performed only for peripheral cells in acini in one focal plane. ...
... Therefore, it is important to nd oxidation substrates contributing to restoration of mitochondrial oxidative capacity of and ATP production increase for acinar cell protection. In our previous study, we have shown that glutamine in moderate concentration (2 mM) prevents mitochondrial membrane depolarization and pancreatic acini necrosis caused by a combination of ethanol and cholecystokinin (Manko et al., 2021). ...
... Alanine can be catabolized to pyruvate by alanine transaminase (McCommis et al. 2015). Pyruvate in vitro enhances the oxidative and ATP-productive capacity of mitochondria and protects pancreatic acinar cells from toxic substances (Manko et al., 2021;Peng et al., 2018). ...
Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acinar cells. Adequate nutritional support is important for acute pancreatitis treatment. However, some amino acids, such as arginine and lysine, are toxic for pancreatic acinar cells in high concentrations. The study aimed to select the candidate amino acids as the best non-toxic energy sources for supplemental therapy of acute pancreatitis.
Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1–20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell necrosis and apoptosis were evaluated with fluorescent microscopy and DNA gel electrophoresis.
Among the tested amino acids, glutamate, glutamine, alanine, lysine and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, while arginine, histidine and asparagine were not. Lysine, arginine and glutamine (20 mM) caused complete necrosis of acinar cells after 24 h of incubation. Glutamine also caused early (2–4 h) cell swelling and blebbing. Aspartate, asparagine and glutamate only moderately increased the number of necrotic cells, while alanine and histidine were not toxic. No significant apoptosis developed after incubation with amino acids.
In conclusion, we propose alanine and glutamate as safe candidate amino acid supplements for patients with acute pancreatitis.
... Chronic ethanol feeding makes the pancreas more susceptible to cerulein-induced pancreatitis [6,7]. Similarly, acute exposure to ethanol in combination with cholecystokinin in vitro or in vivo may cause pancreatic acinar cell death or blebbing [8]. Despite the low clinical relevance of hyperstimulation with cholecystokinin or its analogues, these experiments prove that etha-nol exposure causes the accumulation of defects of some kind in pancreatic acinar cells. ...
Chronic alcohol consumption may cause pancreatitis and alcohol-related liver diseases. Both adaptation and damage of liver mitochondria in animals on chronic ethanol and high-fat diets were demonstrated. It is currently not clear if ethanol or its metabolites such as fatty acid ethyl esters can cause mitochondrial damage to the pancreas. The present study aimed to evaluate the effect of chronic ethanol administration in combination with a high-fat diet on mitochondrial respiration in both pancreatic acinar cells and hepatocytes of rats. Wistar male rats on a high-fat diet (35% calories) were administered ethanol (6 g/kg body weight) by oral gavage for 14 days. Pancreatic acini cells and hepatocytes were isolated with collagenase digestion. The respiration of isolated cells was studied with a Clark electrode. Ethanol administration to rats kept on a high-fat diet was followed by a rapid loss of animal weight during the first 5 days of the experiment and diminished secretory response of pancreatic acini to acetylcholine, however, no changes in acinar cells ultrastructure, basal, oligomycin-insensitive or FCCP-uncoupled respiration were found. Meanwhile ethanol caused a significant (~40%) increase in basal and maximal FCCP-uncoupled respiration rate of isolated hepatocytes. In conclusion, chronic ethanol administration to rats on a high-fat diet does not cause mitochondrial damage in the pancreas, while mitochondria of the liver adapt to ethanol by increasing respiration rate. Keywords: ethanol, hepatocytes, high fat diet, mitochondrial respiration, pancreatic acinar cells
... Using FCCP to enhance mitochondrial HAD(P)H oxidation caused the decrease of its autofluorescence, which did not depend on the diet but was significantly less pronounced when pyruvate was oxidized, indicating increased tricarboxylic cycle activity (Fig. 4, D). This confirms our previous findings that pyruvate enhances mitochondrial functions in isolated rat pancreatic acini [24]. ...
An unhealthy diet often is a cause of obesity, chronic inflammation, and metabolic disruption in multiple organs. However, the direct influence of elevated lipid or sugar consumption on liver, pancreatic, and sperm mitochondria is not well understood. The aim of the study was to investigate the functional activity of mitochondria of liver, pancreatic acinar cells, and sperm cells in rats on a short-term (7 weeks) diet with high fat or high fat and high sugar content. Male Wistar rats were on a basic, high-fat or high-fat high-sugar diet for 7 weeks. At the end of the experiment, visceral fat mass, blood glucose and lipids were measured. Mitochondrial functional activity was evaluated with oxygen consumption assay. In isolated pancreatic acinar cells, NAD(P)H autofluorescence and mitochondrial membrane potential were also studied. No difference in body mass was observed between the 3 groups at the end of the experiment. Visceral fat mass was slightly but significantly elevated in rats on a high-fat high-sugar diet. Both diets did not affect plasma glucose or triglyceride levels but caused a modest elevation of total plasma cholesterol. Respiration and oxidative phosphorylation of isolated liver mitochondria were not affected by any experimental diet. In pancreatic acinar cells, a high-fat diet caused a significant decrease of basal respiration by ~15%, but no effects were observed on the maximal rate of uncoupled respiration, mitochondrial membrane potential, or NAD(P)H autofluorescence. In these cells, a ketone body 3-hydroxybutyrate caused elevation of uncoupled respiration and NAD(P)H level irrespectively of the diet. Diets did not cause any change in sperm concentration, viability or motility. Surprisingly, in animals on a high-fat high-sugar diet, a significant increase in both basal and maximal respiration of sperm cells was observed. Collectively, these data show that while the elevated fat and sugar content in the diet does not cause significant obesity, no detrimental effects on mitochondria of the liver, pancreas, and sperm cells are observed. Keywords: diet, liver, mitochondria, pancreatic acinar cells, sperm
... A number of preclinical studies are aimed at treatment of pancreatitis by restoring the mitochondrial oxidative function (Shore et al. 2016;Mukherjee et al. 2016;Javed et al. 2018). Oxidative substrate pyruvate in vitro enhances the oxidative and ATP-productive capacity of mitochondria and protects pancreatic acinar cells from toxic substances (Peng et al. 2018;Manko et al. 2019Manko et al. , 2021. It is interesting that substantial levels of pyruvate are quickly accumulated in the pancreas among other organs after intravenous administration (Serrao et al. 2018), making this substance a very attractive choice for acute pancreatitis management. ...
Plasma amino acid levels are altered upon many pathological conditions including acute pancreatitis. It is unclear whether amino acids can be used as specific biomarker of acute pancreatitis severity or recovery. Development of acute pancreatitis is associated with mitochondrial dysfunction and decreased cytosolic ATP level. Sodium pyruvate is considered as a potential treatment of pancreatitis due to its ability to sustain mitochondrial oxidative and ATP-productive capacity in vitro. This study investigated the effect of sodium pyruvate on pancreatic morphology and plasma amino acid levels in rats with acute pancreatitis. Acute pancreatitis in rats was induced by administration of l-arginine (5 g/kg) Experimental treatment group received sodium pyruvate (1 g/kg) for 4 days. On day 8 of the experiment, animals were killed, blood was collected and plasma amino acid concentration was determined with high-performance liquid chromatography. Histological examination showed large areas of fibrosis in the pancreas of animals treated with l-arginine irrespectively of sodium pyruvate administration. Sodium pyruvate improved the plasma amino acid levels. Rats with acute pancreatitis had significantly lower levels of most essential and non-essential amino acids and increased glutamate and aspartate in plasma. Administration of sodium pyruvate completely or partially restored the levels of methionine, phenylalanine, tryptophan, leucine, isoleucine, aspartate, asparagine and ornithine levels, while increasing glutamine and serine to levels significantly higher than control. Plasma lysine, alanine, arginine and taurine remained unaffected in all experimental groups. Sodium pyruvate may be considered for use as a maintenance therapy in acute pancreatitis.
Objectives
Amino acids play an essential role in protein synthesis, metabolism and survival of pancreatic acini. Adequate nutritional support is important for acute pancreatitis treatment. However, high concentrations of arginine and lysine may induce acute pancreatitis. The study aimed to identify the most suitable L-amino acids as safe energy sources for pancreatic acinar cells.
Methods
Pancreatic acini were isolated from male Wistar rats. Effects of amino acids (0.1-20 mM) on uncoupled respiration of isolated acini were studied with a Clark electrode. Cell death was evaluated with fluorescent microscopy and DNA gel electrophoresis.
Results
Among the tested amino acids, glutamate, glutamine, alanine, lysine and aspartate were able to stimulate the uncoupled respiration rate of isolated pancreatic acini, while arginine, histidine and asparagine were not. Lysine, arginine and glutamine (20 mM) caused complete loss of plasma membrane integrity of acinar cells after 24 h of incubation. Glutamine also caused early (2–4 h) cell swelling and blebbing. Aspartate, asparagine and glutamate only moderately decreased the number of viable cells, while alanine and histidine were not toxic. DNA fragmentation assay and microscopic analysis of nuclei showed no evidence of apoptosis in cells treated with amino acids.
Conclusions
Alanine and glutamate are safe and effective energy sources for mitochondria of pancreatic acinar cells.
Background. Assessment of cell viability is crucial in cell studies. Testing plasma membrane integrity is a traditional approach of evaluating cell viability. Mitochondrial functional capacity closely correlates with plasma membrane integrity and overall cell health. This study aimed to investigate whether any aspect of mitochondrial adaptive capacity in isolated pancreatic acini is associated with the quality of isolated pancreatic acini preparations, as determined by the dye exclusion method. Materials and Methods. Experiments were carried out on male Wistar rats weighing 250–300 g. A suspension of isolated pancreatic acini was obtained using collagenase. The rate of oxygen consumption of rat isolated pancreatic acini was measured with Clark oxygen electrode. Basal respiration of isolated pancreatic acini was recorded for approximately 2 min. Afterwards, the mitochondrial adaptive capacity was examined using FCCP in concentrations from 0.5 to 2 μM. Uncoupled respiratory stability was calculated as a ratio of respiration rate at high and low FCCP concentrations. Plasma membrane integrity was assessed with trypan blue staining. A total of 74 preparations of isolated pancreatic acini were used in this study. Results. In all experiments, 92–99 % of pancreatic acinar cells exhibited negative trypan blue staining, indicating intact plasma membranes. The basal and maximal uncoupled respiration rates were not affected by the fraction of trypan-negative cells. However, acini preparations with <less than 95 % plasma membrane integrity had significantly lower uncoupled respiration rates when exposed to a high concentration of FCCP (2 µM), indicating reduced stability of uncoupled respiration. Conclusions. Results of the study suggest that the stability of uncoupled respiration can serve as a novel metabolic functional test to complement the existing methods for assessing cell vitality.
Plasma amino acid levels are altered upon many pathological conditions including acute pancreatitis. It is unclear whether amino acids can be used as specific biomarker of acute pancreatitis severity or recovery. Development of acute pancreatitis is associated with mitochondrial dysfunction and decreased cytosolic ATP level. Sodium pyruvate is considered as a potential treatment of pancreatitis due to its ability to sustain mitochondrial oxidative and ATP-productive capacity in vitro. In this study investigated the effect of sodium pyruvate on pancreatic morphology and plasma amino acid levels in rats with acute pancreatitis.
Acute pancreatitis in rats was induced by administration of L-arginine (5 g / kg) and confirmed with histological examination of pancreas. Experimental treatment group received sodium pyruvate (1 g / kg) for 4 days. Blood was collected on day 8 of the experiment and plasma amino acids concentration was determined with high-performance liquid chromatography.
Sodium pyruvate administration did not improve the pancreatic morphology and ultrastructure, but improves the plasma amino acid levels. Rats with acute pancreatitis had significantly lower levels of most essential and non-essential amino acids and increased glutamate and aspartate in plasma. Administration of sodium pyruvate completely or partially restored levels of methionine, phenylalanine, tryptophan, leucine, isoleucine, aspartate, asparagine and ornithine levels, while increasing glutamine and serine to levels significantly higher than control. Plasma lysine, alanine, arginine and taurine remained unaffected remained unaffected in all experimental groups. Sodium pyruvate may be considered for use as a maintenance therapy in acute pancreatitis.
The dependence of uncoupled respiratory capacity of intact pancreatic acini on oxidative substrate supply and functional cell state has not yet been studied in detail. In this study, the respiratory responses of isolated pancreatic acini to FCCP were measured with Clark electrode and mitochondrial membrane potential was assessed with rhodamine123 fluorescence. The response of acini to FCCP was characterized with maximal uncoupled respiration rate, optimal FCCP concentration, respiration acceleration and deceleration. Maximal uncoupled respiration rate substantially increased upon the oxidation of glucose + glutamine (3.03 ± 0.54 r.u.), glucose + glutamine + pyruvate (2.82 ± 0.51 r.u.), glucose + isocitrate (2.71 ± 0.33 r.u.), glucose + malate (2.75 ± 0.38 r.u.), glucose + monomethyl-succinate (2.64 ± 0.42 r.u.) or glucose + dimethyl-α-ketoglutarate (2.36 ± 0.33 r.u.) comparing to glucose alone (1.73–2.02 r.u.) or no substrate (1.76 ± 0.33 r.u.). The optimal FCCP concentration was the highest (1.75 μM) upon glucose + glutamine + pyruvate combination and the lowest (0.5 μM) upon glutamate, combinations of glucose with isocitrate, malate, succinate or α-ketoglutarate. Respiration acceleration after FCCP application was the highest with dimethyl-α-ketoglutarate. Following the peak respiration, time-dependent deceleration was observed. It increased with FCCP concentration and depended on oxidative substrate type. Deceleration was the highest upon malate or isocitrate oxidation but was not observed in case of glutamine or dimethyl-α-ketoglutarate oxidation. Pyruvate alone or in combination with glutamine and glucose significantly decreased the depolarizing effect of FCCP on mitochondrial membrane potential and increased respiration elasticity coefficient with respect to the membrane potential change. Thus, in pancreatic acinar cells, the combination of pyruvate, glutamine and glucose enables the optimal adaptive respiratory response to membrane depolarization.
Acute pancreatitis (AP), a human disease in which the pancreas digests itself, has substantial mortality with no specific therapy. The major causes of AP are alcohol abuse and gallstone complications, but it also occurs as an important side effect of the standard Asparaginase-based therapy for childhood acute lymphoblastic leukaemia. Previous investigations into the mechanisms underlying pancreatic acinar cell death induced by alcohol metabolites, bile acids or Asparaginase indicated that loss of intracellular ATP generation is a significant factor. In isolated mouse pancreatic acinar cells or cell clusters, we now report that removal of extracellular glucose had little effect on this ATP loss, suggesting that glucose metabolism was severely inhibited under these conditions. Surprisingly, we show that replacing glucose with galactose prevented or markedly reduced the loss of ATP and any subsequent necrosis. Addition of pyruvate had a similar protective effect. We also studied the effect of galactose in vivo in mouse models of AP induced either by a combination of fatty acids and ethanol or Asparaginase. In both cases, galactose markedly reduced acinar necrosis and inflammation. Based on these data we suggest that galactose feeding may be used to protect against AP.
The bioenergetics and molecular determinants of the metabolic response to mitochondrial dysfunction are incompletely understood, in part due to a lack of appropriate isogenic cellular models of primary mitochondrial defects. Here, we capitalize on a recently developed cell model with defined levels of m.8993T>G mutation heteroplasmy, mTUNE, to investigate the metabolic underpinnings of mitochondrial dysfunction. We found that impaired utilization of reduced nicotinamide adenine dinucleotide (NADH) by the mitochondrial respiratory chain leads to cytosolic reductive carboxylation of glutamine as a new mechanism for cytosol-confined NADH recycling supported by malate dehydrogenase 1 (MDH1). We also observed that increased glycolysis in cells with mitochondrial dysfunction is associated with increased cell migration in an MDH1-dependent fashion. Our results describe a novel link between glycolysis and mitochondrial dysfunction mediated by reductive carboxylation of glutamine.
Objectives:
Mitochondrial permeability transition pore inhibition is a promising approach to treat acute pancreatitis (AP). We sought to determine (i) the effects of the mitochondrial permeability transition pore inhibitor 3,5-seco-4-nor-cholestan-5-one oxime-3-ol (TRO40303) on murine and human pancreatic acinar cell (PAC) injury induced by fatty acid ethyl esters (FAEEs) or taurolithocholic acid-3-sulfate and (ii) TRO40303 pharmacokinetics and efficacy in experimental alcoholic AP (FAEE-AP).
Methods:
Changes in mitochondrial membrane potential ([INCREMENT]ψm), cytosolic Ca ([Ca]c), and cell fate were examined in freshly isolated murine or human PACs by confocal microscopy. TRO40303 pharmacokinetics were assessed in cerulein-induced AP and therapeutic efficacy in FAEE-AP induced with palmitoleic acid and ethanol. Severity of AP was assessed by standard biomarkers and blinded histopathology.
Results:
TRO40303 prevented loss of [INCREMENT]ψm and necrosis induced by 100 μM palmitoleic acid ethyl ester or 500 μM taurolithocholic acid-3-sulfate in murine and human PACs. Pharmacokinetic analysis found TRO40303 accumulated in the pancreas. A single dose of 3 mg/kg TRO40303 significantly reduced serum amylase (P = 0.043), pancreatic trypsin (P = 0.018), and histopathology scores (P = 0.0058) in FAEE-AP.
Conclusions:
TRO40303 protects mitochondria and prevents necrotic cell death pathway activation in murine and human PACs, ameliorates the severity of FAEE-AP, and is a candidate drug for human AP.This is an open-access article distributed under the terms of the Creative Commons Attribution-Non Commercial-No Derivatives License 4.0 (CCBY-NC-ND), where it is permissible to download and share the work provided it is properly cited. The work cannot be changed in any way or used commercially without permission from the journal.
The dependence of respiration rate of rat permeabilized acinar pancreacytes on oxidative substrates concentration was studied at various [Ca2+] - 10-8-10-6 M. Pancreacytes were permeabilized with 50 microg of digitonin per 1 million cells. Respiration rate was measured polarographically using the Clark electrode at oxidation of succinate or pyruvate either glutamate in the presence of malate. Parameters of Michaelis-Menten equation were calculated by the method of Cornish-Bowden or using Idi-Hofsti coordinates and parameters of Hill equation - using coordinates {v; v/[S]h}. In the studied range of [Ca2+] the kinetic dependence of respiration at pyruvate oxidation is described by the Michaelis-Menten equation, and at oxidation of succinate or glutamate - by Hill equation with h = 1.11-1.43 and 0.50-0.85, respectively. The apparent constant of respiration half-activation (K0.5) did not significantly change in the studied range of [Ca2+] while at 10-7 M Ca2+ it was 0.90 +/- 0.06 mM for succinate, 0.096 +/- 0.007 mM for pyruvate and 0.34 +/- 0.03 mM for glutamate. Maximum respiration rate Vax at pyruvate oxidation increased from 0.077 +/- 0.002 to 0.119 +/- 0.002 and 0.140 +/- 0.002 nmol O2/(s.million cells) due to the increase of [Ca2+] from 10-7 to 5x10-7 or 10-6 M, respectively. At oxidation of succinate or glutamate Ca2+ did not significantly affect Vmax Thus, the increase of [Ca2+] stimulates respiration of mitochondria in situ of acinar pancreacytes at oxidation of exogenous pyruvate (obviously due to pyruvate dehydrogenase activation), but not at succinate or glutamate oxidation.
Acute pancreatitis is caused by toxins that induce acinar cell calcium overload, zymogen activation, cytokine release and cell death, yet is without specific drug therapy. Mitochondrial dysfunction has been implicated but the mechanism not established.
We investigated the mechanism of induction and consequences of the mitochondrial permeability transition pore (MPTP) in the pancreas using cell biological methods including confocal microscopy, patch clamp technology and multiple clinically representative disease models. Effects of genetic and pharmacological inhibition of the MPTP were examined in isolated murine and human pancreatic acinar cells, and in hyperstimulation, bile acid, alcoholic and choline-deficient, ethionine-supplemented acute pancreatitis.
MPTP opening was mediated by toxin-induced inositol trisphosphate and ryanodine receptor calcium channel release, and resulted in diminished ATP production, leading to impaired calcium clearance, defective autophagy, zymogen activation, cytokine production, phosphoglycerate mutase 5 activation and necrosis, which was prevented by intracellular ATP supplementation. When MPTP opening was inhibited genetically or pharmacologically, all biochemical, immunological and histopathological responses of acute pancreatitis in all four models were reduced or abolished.
This work demonstrates the mechanism and consequences of MPTP opening to be fundamental to multiple forms of acute pancreatitis and validates the MPTP as a drug target for this disease.
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Acute pancreatitis is a serious and sometimes fatal inflammatory disease where the pancreas digests itself. The non-oxidative
ethanol metabolites palmitoleic acid (POA) and POA-ethylester (POAEE) are reported to induce pancreatitis caused by impaired
mitochondrial metabolism, cytosolic Ca2+ ([Ca2+]i) overload and necrosis of pancreatic acinar cells. Metabolism and [Ca2+]i are linked critically by the ATP-driven plasma membrane Ca2+-ATPase (PMCA) important for maintaining low resting [Ca2+]i. The aim of the current study was to test the protective effects of insulin on cellular injury induced by the pancreatitis-inducing
agents, ethanol, POA, and POAEE. Rat pancreatic acinar cells were isolated by collagenase digestion and [Ca2+]i was measured by fura-2 imaging. An in situ [Ca2+]i clearance assay was used to assess PMCA activity. Magnesium green (MgGreen) and a luciferase-based ATP kit were used to assess
cellular ATP depletion. Ethanol (100 mm) and POAEE (100 μm) induced a small but irreversible Ca2+ overload response but had no significant effect on PMCA activity. POA (50–100 μm) induced a robust Ca2+ overload, ATP depletion, inhibited PMCA activity, and consequently induced necrosis. Insulin pretreatment (100 nm for 30 min) prevented the POA-induced Ca2+ overload, ATP depletion, inhibition of the PMCA, and necrosis. Moreover, the insulin-mediated protection of the POA-induced
Ca2+ overload was partially prevented by the phosphoinositide-3-kinase (PI3K) inhibitor, LY294002. These data provide the first
evidence that insulin directly protects pancreatic acinar cell injury induced by bona fide pancreatitis-inducing agents, such as POA. This may have important therapeutic implications for the treatment of pancreatitis.
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.
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.
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.
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.
Reductively metabolized glutamine is a major cellular carbon source for fatty acid synthesis during hypoxia or when mitochondrial respiration is impaired. Yet, a mechanistic understanding of what determines reductive metabolism is missing. Here we identify several cellular conditions where the α-ketoglutarate/citrate ratio is changed due to an altered acetyl-CoA to citrate conversion, and demonstrate that reductive glutamine metabolism is initiated in response to perturbations that result in an increase in the α-ketoglutarate/citrate ratio. Thus, targeting reductive glutamine conversion for a therapeutic benefit might require distinct modulations of metabolite concentrations rather than targeting the upstream signalling, which only indirectly affects the process.
Fluorescent tracer dyes represent an important class of sub-cellular probes and allow the examination of cellular processes in real-time with minimal impact upon these processes. Such tracer dyes are becoming increasingly used for the examination of membrane transport processes, as they are easy-to-use, cost effective probe substrates for a number of membrane protein transporters. Rhodamine 123, a member of the rhodamine family of flurone dyes, has been used to examine membrane transport by the ABCB1 gene product, MDR1. MDR1 is viewed as the archetypal drug transport protein, and is able to efflux a large number of clinically relevant drugs. In addition, ectopic activity of MDR1 has been associated with the development of multiple drug resistance phenotype, which results in a poor patient response to therapeutic intervention. It is thus important to be able to examine the potential for novel compounds to be MDR1 substrates. Given the increasing use rhodamine 123 as a tracer dye for MDR1, a full characterisation of its spectral properties in a range of in vitro assay-relevant media is warranted. Herein, we determine λmax for excitation and emission or rhodamine 123 and its metabolite rhodamine 110 in commonly used solvents and extraction buffers, demonstrating that fluorescence is highly dependent on the chemical environment: Optimal parameters are 1% (v/v) methanol in HBSS, with λex = 505 nm, λem = 525 nm. We characterise the uptake of rhodamine 123 into cells, via both passive and active processes, and demonstrate that this occurs primarily through OATP1A2-mediated facilitated transport at concentrations below 2 µM, and via micelle-mediated passive diffusion above this. Finally, we quantify the intracellular sequestration and metabolism of rhodamine 123, demonstrating that these are both cell line-dependent factors that may influence the interpretation of transport assays.
The gastrointestinal hormone CCK exists in various molecular forms, with differences in bioactivity between the well-characterized CCK-8 and larger CCK-58 previously reported. We have compared the effects of these peptides on cytosolic calcium concentration ([Ca(2+)](c)), mitochondrial metabolism, enzyme secretion, and cell fate in murine isolated pancreatic acinar cells using fluorescence confocal microscopy and patch-clamp electrophysiology. CCK-58 (1-10 pM) induced transient, oscillatory increases of [Ca(2+)](c), which showed apical to basolateral progression and were associated with a rise of mitochondrial NAD(P)H. CCK-58 (10 pM) induced zymogen exocytosis in isolated cells and amylase secretion from isolated cells and whole tissues. Hyperstimulation with supraphysiological CCK-58 (5 nM) induced a single large increase of [Ca(2+)](c) that declined to a plateau, which remained above the basal level 20 min after application and was dependent on external Ca(2+) entry. In cells dispersed from the same tissues, CCK-8 induced similar patterns of responses to those of CCK-58, with oscillatory increases of [Ca(2+)](c) at lower (pM) concentrations and sustained responses at 5 nM. CCK-58 and CCK-8 exhibited similar profiles of action on cell death, with increases in necrosis at high CCK-58 and CCK-8 (10 nM) that were not significantly different between peptides. The present experiments indicate that CCK-8 and CCK-58 have essentially identical actions on the acinar cell at high and low agonist concentrations, suggesting an action via the same receptor and that the differences observed in an intact rat model may result from indirect effects of the peptides. Our data strengthen the argument that CCK-58 is an important physiological form of this gastrointestinal hormone.
Apoptosis and necrosis are critical parameters of pancreatitis, the mechanisms of which remain unknown. Many characteristics of pancreatitis can be studied in vitro in pancreatic acini treated with high doses of cholecystokinin (CCK). We show here that CCK stimulates apoptosis and death signaling pathways in rat pancreatic acinar cells, including caspase activation, cytochrome c release, and mitochondrial depolarization. The mitochondrial dysfunction is mediated by upstream caspases (possibly caspase-8) and, in turn, leads to activation of caspase-3. CCK causes mitochondrial alterations through both permeability transition pore-dependent (cytochrome c release) and permeability transition pore-independent (mitochondrial depolarization) mechanisms. Caspase activation and mitochondrial alterations also occur in untreated pancreatic acinar cells; however, the underlying mechanisms are different. In particular, caspases protect untreated acinar cells from mitochondrial damage. We found that caspases not only mediate apoptosis but also regulate other parameters of CCK-induced acinar cell injury that are characteristic of pancreatitis; in particular, caspases negatively regulate necrosis and trypsin activation in acinar cells. The results suggest that the observed signaling pathways regulate parenchymal cell injury and death in CCK-induced pancreatitis. Protection against necrosis and trypsin activation by caspases can explain why the severity of pancreatitis in experimental models correlates inversely with the extent of apoptosis.
In this study, we investigated the effects of secretagogues and bile acids on the mitochondrial membrane potential of pancreatic acinar cells. We measured the mitochondrial membrane potential using the tetramethylrhodamine-based probes tetramethylrhodamine ethyl ester and tetramethylrhodamine methyl ester. At low levels of loading, these indicators appeared to have a low sensitivity to the uncoupler carbonyl cyanide m-chlorophenylhydrazone, and no response was observed to even high doses of cholecystokinin. When loaded at high concentrations, tetramethylrhodamine methyl ester and tetramethylrhodamine ethyl ester undergo quenching and can be dequenched by mitochondrial depolarization. We found the dequench mode to be 2 orders of magnitude more sensitive than the low concentration mode. Using the dequench mode, we resolved mitochondrial depolarizations produced by supramaximal and by physiological concentrations of cholecystokinin. Other calcium-releasing agonists, acetylcholine, JMV-180, and bombesin, also produced mitochondrial depolarization. Secretin, which employs the cAMP pathway, had no effect on the mitochondrial potential; dibutyryl cAMP was also ineffective. The cholecystokinin-induced mitochondrial depolarizations were abolished by buffering cytosolic calcium. A non-agonist-dependent calcium elevation induced by thapsigargin depolarized the mitochondria. These experiments suggest that a cytosolic calcium concentration rise is sufficient for mitochondrial depolarization and that the depolarizing effect of cholecystokinin is mediated by a cytosolic calcium rise. Bile acids are considered possible triggers of acute pancreatitis. The bile acids taurolithocholic acid 3-sulfate, taurodeoxycholic acid, and taurochenodeoxycholic acid, at low submillimolar concentrations, induced mitochondrial depolarization, resolved by the dequench mode. Our experiments demonstrate that physiological concentrations of secretagogues and pathologically relevant concentrations of bile acids trigger mitochondrial depolarization in pancreatic acinar cells.
Supramaximal stimulation of isolated pancreatic acini with specific agonists such as CCK induces the formation of large basolateral blebs, redistributes filamentous actin, and inhibits secretion. Rho family small G proteins are well documented for their function in actin reorganization that determines cell shape and have been suggested to play a role in secretion. Here, we determined whether Rho and Rac are involved in the morphological changes, actin redistribution, and inhibition of amylase secretion induced by high concentrations of CCK. Introduction of constitutively active RhoV14 and RacV12 but not Cdc42V12 in mouse pancreatic acini by adenoviral vectors stimulated acinar morphological changes including basolateral protrusions, increased the total amount of F-actin, and reorganized the actin cytoskeleton. Dominant-negative RhoN19, Clostridium botulinum C3 exotoxin, which inhibits Rho, and dominant-negative RacN17 all partially blocked CCK-induced acinar morphological changes and actin redistribution. To study the correlation between actin polymerization and acinar shape changes, two marine toxins were employed. Jasplakinolide, a reagent that facilitates actin polymerization and stabilizes F-actin, stimulated acinar basolateral protrusions, whereas latrunculin, which sequesters actin monomers, blocked CCK-induced acinar blebbing. Unexpectedly, RhoV14, RacV12, and jasplakinolide all increased amylase secretion by CCK from 30 pM to 10 nM. The data suggest that Rho and Rac are involved in CCK-evoked changes in acinar morphology, actin redistribution, and secretion and that inhibition of secretion by high concentrations of CCK is not directly coupled to the changes in acinar morphology.
Anoikis is a programmed cell death induced by loss of anchorage that is involved in tissue homeostasis and disease. Ethanol is an important teratogen that induces marked central nervous system (CNS) dysfunctions. Here we show that astrocytes exposed to ethanol undergo morphological changes associated with anoikis, including the peripheral reorganization of both focal adhesions and actin-myosin system, cell contraction, membrane blebbing and chromatin condensation. We found that either the small GTPase RhoA or its effector ROCK-I (Rho kinase), promotes membrane blebbing in astrocytes. Ethanol induces a ROCK-I activation that is mediated by RhoA, rather than by caspase-3 cleavage. Accordingly, the RhoA inhibitor C3, completely abolishes the ethanol-induced ROCK-I activation. Furthermore, inhibition of both RhoA and ROCK prevents the membrane blebbing induced by ethanol. Ethanol also promotes myosin light chain (MLC) phosphorylation, which might be involved in the actin-myosin contraction. All of these findings strongly support that ethanol-exposed astrocytes undergo apoptosis by anoikis and also that the RhoA/ROCK-I/MLC pathway participates in this process.
Pancreatitis is an increasingly common disease that carries a significant mortality and which lacks specific therapy. Pathological calcium signalling is an important contributor to the initiating cell injury, caused by or acting through mitochondrial inhibition. A principal effect of disordered cell signalling and impaired mitochondrial function is cell death, either by apoptosis that is primarily protective, or by necrosis that is deleterious, both locally and systemically. Mitochondrial calcium overload is particularly important in necrotic injury, which may include damage mediated by the mitochondrial permeability transition pore. The role of reactive oxygen species remains controversial. Present understanding of the part played by disordered pancreatic acinar calcium signalling and mitochondrial inhibition offers several new potential therapeutic targets.
Suprastimulation of pancreatic acinar cells with specific agonists inhibits zymogen secretion and induces the formation of large basolateral blebs. Currently the molecular mechanisms that mediate this dramatic morphologic response are undefined. Further, it is unclear if blebbing represents a terminal or reversible event. Using computer-enhanced video microscopy of living acini we have found that these large blebs form rapidly (within 2-3 minutes) and exhibit ameboid undulations. They are induced by small increases in agonist concentration and require an energy-dependent phosphorylation event. Remarkably, the blebs are rapidly absorbed when agonist levels are reduced, indicating that blebbing is a reversible response to a physiological stimulus, not a terminal event. Morphological methods show that these dramatic changes in cell shape are accompanied by a marked reorganization of actin and myosin II at the basolateral domain. During 30 minutes of suprastimulation, both basolateral actin and myosin II gradually increase to form a ring centered at the necks of the blebs. Immunocytochemical and biochemical studies with a phospho-specific antibody to the myosin regulatory light chain reveal an activation of myosin II in suprastimulated acini that is completely absent in resting cells. Studies using cytoskeletal antagonistic drugs indicate that bleb formation and motility require actin remodeling concomitant with an activation of myosin II. This aberrant activation and reorganization of the actin-myosin cytoskeleton is likely to have detrimental effects on acinar cell function. Additionally, this mechanism of bleb formation may be conserved among other forms of physiological blebbing events.
Intrapancreatic trypsin activation by dysregulated macroautophagy/autophagy and pathological exocytosis of zymogen granules (ZGs), along with activation of inhibitor of NFKB/NF-κB kinase (IKK) are necessary early cellular events in pancreatitis. How these three pancreatitis events are linked is unclear. We investigated how SNAP23 orchestrates these events leading to pancreatic acinar injury. SNAP23 depletion was by knockdown (SNAP23-KD) effected by adenovirus-shRNA (Ad-SNAP23-shRNA/mCherry) treatment of rodent and human pancreatic slices and in vivo by infusion into rat pancreatic duct. In vitro pancreatitis induction by supraphysiological cholecystokinin (CCK) or ethanol plus low-dose CCK were used to assess SNAP23-KD effects on exocytosis and autophagy. Pancreatitis stimuli resulted in SNAP23 translocation from its native location at the plasma membrane to autophagosomes, where SNAP23 would bind and regulate STX17 (syntaxin17) SNARE complex-mediated autophagosome-lysosome fusion. This SNAP23 relocation was attributed to IKBKB/IKKβ-mediated SNAP23 phosphorylation at Ser95 Ser120 in rat and Ser120 in human, which was blocked by IKBKB/IKKβ inhibitors, and confirmed by the inability of IKBKB/IKKβ phosphorylation-disabled SNAP23 mutant (Ser95A Ser120A) to bind STX17 SNARE complex. SNAP23-KD impaired the assembly of STX4-driven basolateral exocytotic SNARE complex and STX17-driven SNARE complex, causing respective reduction of basolateral exocytosis of ZGs and autolysosome formation, with consequent reduction in trypsinogen activation in both compartments. Consequently, pancreatic SNAP23-KD rats were protected from caerulein and alcoholic pancreatitis. This study revealed the roles of SNAP23 in mediating pathological basolateral exocytosis and IKBKB/IKKβ’s involvement in autolysosome formation, both where trypsinogen activation would occur to cause pancreatitis. SNAP23 is a strong candidate to target for pancreatitis therapy.
Background & aims:
Little is known about the signaling pathways that initiate and promote acute pancreatitis (AP). The pathogenesis of AP has been associated with abnormal increases in cytosolic Ca(2+), mitochondrial dysfunction, impaired autophagy, and endoplasmic reticulum (ER) stress. We analyzed the mechanisms of these dysfunctions and their relationships, and how these contribute to development of AP in mice and rats.
Methods:
Pancreatitis was induced in C57BL/6J mice (control) and mice deficient in peptidylprolyl isomerase D (cyclophilin D, encoded by Ppid) by administration of L-arginine (also in rats), caerulein, bile acid, or an AP-inducing diet. Parameters of pancreatitis, mitochondrial function, autophagy, ER stress, and lipid metabolism were measured in pancreatic tissue, acinar cells, and isolated mitochondria. Some mice with AP were given trehalose to enhance autophagic efficiency. Human pancreatitis tissues were analyzed by immunofluorescence.
Results:
Mitochondrial dysfunction in pancreas of mice with AP was induced by either mitochondrial Ca(2+) overload or through a Ca(2+) overload-independent pathway that involved reduced activity of F-ATP synthase (80% inhibition in pancreatic mitochondria isolated from rats or mice given L-arginine). Both pathways were mediated by cyclophilin D and led to mitochondrial depolarization and fragmentation. Mitochondrial dysfunction caused pancreatic ER stress, impaired autophagy, and deregulation of lipid metabolism. These pathologic responses were abrogated in cyclophilin D-knockout mice. Administration of trehalose largely prevented trypsinogen activation, necrosis, and other parameters of pancreatic injury in mice with L-arginine AP. Tissues from patients with pancreatitis had markers of mitochondrial damage and impaired autophagy, compared with normal pancreas.
Conclusions:
In different animal models, we find a central role for mitochondrial dysfunction, and for impaired autophagy as its principal downstream effector, in development of AP. In particular, the pathway involving enhanced interaction of cyclophilin D with F-ATP synthase mediates L-arginine induced pancreatitis, a model of severe AP the pathogenesis of which has remained unknown. Strategies to restore mitochondrial and/or autophagic function might be developed for treatment of AP.
Knowledge of the molecular mechanisms of acute pancreatitis is largely based on studies using rodents. To assess similar mechanisms in humans, we performed ex vivo pancreatitis studies in human acini isolated from cadaveric pancreata from organ donors. Since data on these human acinar preparations are sparse, we assessed their functional integrity, and cellular and organellar morphology using light, fluorescence and electron microscopy; and their proteome by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Acinar cell responses to the muscarinic agonist carbachol (CCh) and the bile acid taurolithocholic acid-3-sulfate (TLCS) were also analyzed. Proteomic analysis of acini from donors of diverse ethnicity showed similar profiles of digestive enzymes and proteins involved in translation, secretion and endolysosomal function. Human acini preferentially expressed the muscarinic acetylcholine receptor M3 and maintained physiologic responses to CCh for at least 20 h. As in rodent acini, human acini exposed to toxic concentrations of CCh and TLCS responded with trypsinogen activation, decreased cell viability, organelle damage manifest by mitochondrial depolarization, disordered autophagy and pathological endoplasmic reticulum stress. Human acini also secreted inflammatory mediators elevated in acute pancreatitis patients including IL6, TNFα, IL1β, CCL2, CCL3, MIF, and chemokines mediating neutrophil and monocyte infiltration. In conclusion, human cadaveric pancreatic acini maintain physiologic functions and have similar pathological responses and organellar disorders with pancreatitis-causing treatments as observed in rodent acini.
Opening of the mitochondrial permeability transition pore (MPTP) causes mitochondrial dysfunction and necrosis in acute pancreatitis (AP), a condition without specific drug treatment. Cyclophilin D (CypD) is a mitochondrial matrix peptidyl-prolyl isomerase that regulates the MPTP and is a drug target for AP. We have synthesised urea-based small molecule inhibitors of cyclophilins and tested them against CypD using binding and isomerase activity assays. Thermodynamic profiles of the CypD/inhibitor interactions were determined by isothermal titration calorimetry. Seven new high-resolution crystal structures of CypD-inhibitor complexes were obtained to guide optimisation. Compounds 4, 13, 14 and 19 were tested in freshly isolated murine pancreatic acinar cells (PACs) to determine inhibition of toxin-induced loss of mitochondrial membrane potential (∆Ψm) and necrotic cell death pathway activation. Compound 19 was found to have a Kd of 410 nM and favourable thermodynamic profile, showed significant protection of ∆Ψm and reduced necrosis of murine as well as human PACs. Compound 19 holds significant promise for future lead optimisation.
Aim:
Acetylcholine as one of the main secretagogues modulates mitochondrial functions in acinar pancreacytes, presumably due to increase in ATP hydrolysis or Ca(2+) transport into mitochondria. The aim of this work was to investigate the mechanisms of carbachol (CCh) action on respiration and oxidative phosphorylation of isolated pancreatic acini.
Methods:
Respiration of intact or permeabilized rat pancreatic acini was studied at 37 °C using a Clark oxygen electrode.
Results:
Respiration rate of isolated acini in rest was 0.27 ± 0.01 nmol O2 s(-1) 10(-6) cells. Addition of 10 μM CCh into respiration chamber evoked biphasic stimulation of respiration. Rapid increase of respiration by 20.1% lasted for approx. 1 min, followed by decrease to level by 11.5% higher than control. Addition of 1 μm CCh caused monophasic increase by 11.5%. Preincubation (5 min) with 1 or 10 μm CCh elevated respiration rate by 12.5 or 11.2% respectively. FCCP prevented the effect of CCh. Preincubation with 1 (but not 10) μm CCh increased FCCP-uncoupled respiration rate. Thapsigargin slightly elevated respiration, but ryanodine did not. Application of 2-aminoethoxydiphenyl borate or ruthenium red prevented the effects of CCh on respiration, while oligomycin abolished them. Preincubation with 1 μm CCh prior to cell permeabilization increased respiration rate at pyruvate+malate oxidation, but not at succinate oxidation. In contrast, preincubation with 10 μm CCh decreased pyruvate+malate oxidation.
Conclusion:
Medium CCh dose (1 μm) intensifies respiration and oxidative phosphorylation of acinar pancreacytes by feedforward mechanism via Ca(2+) transport into mitochondria and activation of Ca(2+) /ADP-sensitive mitochondrial dehydrogenases. Prolonged action of high CCh dose (10 μm) might impair mitochondrial functions.
Background & aims:
Opening of the mitochondrial permeability transition pore (MPTP) causes loss of the mitochondrial membrane potential (ΔΨm) and, ultimately, adenosine triphosphate depletion and necrosis. Cells deficient in cyclophilin D (CypD), a component of the MPTP, are resistant to MPTP opening, loss of ΔΨm, and necrosis. Alcohol abuse is a major risk factor for pancreatitis and is believed to sensitize the pancreas to stressors, by poorly understood mechanisms. We investigated the effects of ethanol on the pancreatic MPTP, the mechanisms of these effects, and their role in pancreatitis.
Methods:
We measured ΔΨm in mouse pancreatic acinar cells incubated with ethanol alone and in combination with physiologic and pathologic concentrations of cholecystokinin-8 (CCK). To examine the role of MPTP, we used ex vivo and in vivo models of pancreatitis, induced in wild-type and CypD(-/-) mice by a combination of ethanol and CCK.
Results:
Ethanol reduced basal ΔΨm and converted a transient depolarization, induced by physiologic concentrations of CCK, into a sustained decrease in ΔΨm, resulting in reduced cellular adenosine triphosphate and increased necrosis. The effects of ethanol and CCK were mediated by MPTP because they were not observed in CypD(-/-) acinar cells. Ethanol and CCK activated MPTP through different mechanisms-ethanol by reducing the ratio of oxidized nicotinamide adenine dinucleotide to reduced nicotinamide adenine dinucleotide, as a result of oxidative metabolism, and CCK by increasing cytosolic Ca(2+). CypD(-/-) mice developed a less-severe form of pancreatitis after administration of ethanol and CCK.
Conclusions:
Oxidative metabolism of ethanol sensitizes pancreatic mitochondria to activate MPTP, leading to mitochondrial failure; this makes the pancreas susceptible to necrotizing pancreatitis.
Mitochondria maintain numerous energy-consuming processes in pancreatic acinar cells, yet characteristics of pancreatic mitochondrial oxidative phosphorylation in native conditions are poorly studied. Besides, it is not known which type of solution is most adequate to preserve functions of pancreatic mitochondria in situ. Here we propose a novel experimental protocol suitable for in situ analysis of pancreatic mitochondria metabolic states. Isolated rat pancreatic acini were permeabilized with low doses of digitonin. Different metabolic states of mitochondria were examined in KCl- and sucrose-based solutions using Clark oxygen electrode. Respiration of digitonin-treated, unlike of intact, acini was substantially intensified by succinate or mixture of pyruvate plus malate. Substrate-stimulated respiration rate did not depend on solution composition. In sucrose-based solution, oligomycin inhibited State 3 respiration at succinate oxidation by 65.4% and at pyruvate plus malate oxidation by 60.2%, whereas in KCl-based solution, by 32.0% and 36.1%, respectively. Apparent respiratory control indices were considerably higher in sucrose-based solution. Rotenone or thenoyltrifluoroacetone severely inhibited respiration, stimulated by pyruvate plus malate or succinate, respectively. This revealed low levels of non-mitochondrial oxygen consumption of permeabilized acinar cells. These results suggest a stronger coupling between respiration and oxidative phosphorylation in sucrose-based solution. Copyright © 2012 John Wiley & Sons, Ltd.
A new alcohol oxidase-peroxidase method of ethanol assay in fermented musts and wine products is described and compared to
conventional methods routinely used in winemaking. The sensitivity, accuracy, and reliability of this method were determined.
The results of ethanol determination in fermented musts and wines correlated well with the data obtained by refractometry
(correlation coefficient R = 0.9595, p < 0.0001) and densitometry (correlation coefficient R = 0.9384, p < 0.0001). The proposed method is less time- and labor-consuming and allows simultaneous analysis of a series of wine samples.
Alcohols are widely used as industrial solvents and chemical intermediates but can cause serious damage to human health. Nevertheless, few studies have addressed the molecular mechanisms underlying the cytotoxicity of industrial alcohols, with the notable exception of ethanol. The goal of our current study is to elucidate the molecular mechanism of cytotoxicity caused by primary alcohols containing longer carbon chains than ethanol. We find that 1-butanol induces morphological changes in H9c2 cardiomyoblastoma including nuclear condensation and membrane blebbing, both of which are features of apoptotic response. Moreover, a decrease in the mitochondrial membrane potential, the cytosolic release of cytochrome c, and the activation of caspase 9 and 3 was observed, thus revealing the activation of the mitochondrial apoptotic pathway by 1-butanol. The addition of Y-27632, a specific inhibitor of Rho-associated kinase (ROCK), suppressed the membrane blebbing and mitochondrial apoptotic pathway. In comparison z-VAD-fmk, a pan-caspase inhibitor, did not inhibit membrane blebbing but did prevent cell death following exposure to 1-butanol. These results indicate that mitochondrial pathway of apoptosis and membrane blebbing are parallel phenomena that occur downstream of ROCK. This kinase thus plays an essential role in 1-butanol cytotoxicity and subsequent cell death in H9c2 cells.
Phosphatidylethanol (PEth) is a group of alcohol-modified phospholipids present in cell membranes after heavy drinking. Our aim was to demonstrate the presence of human plasma antibodies binding to PEth and to address their specificity and value in detecting subjects engaged in heavy alcohol consumption. Antibodies to PEth were analyzed in plasma from heavy drinkers (n = 20), patients with alcoholic pancreatitis (n = 58) and control subjects (n = 24), using chemiluminescent immunoassay. Heavy drinkers and patients with alcoholic pancreatitis demonstrated significantly lower levels of plasma IgG, IgA and IgM titers to PEth compared with controls (P < 0.001). The specificity of the antibodies to PEth was demonstrated with competitive liquid phase immunoassays and flow cytometry. The plasma IgG, but not IgA or IgM, titers to PEth in heavy drinkers correlated with the whole blood PEth concentration determined by liquid chromatography-mass spectrometry (r = 0.655, P = 0.002). Compared with traditional markers for alcohol abuse (aspartate aminotransferase, gamma-glutamyl transpeptidase and mean corpuscular volume), receiver operating characteristic curve analysis showed that a low plasma IgA to PEth had the highest area under the curve (AUC 0.940, P < 0.001). In conclusion, plasma IgG, IgA and IgM antibodies binding specifically to PEth were found in subjects of all study groups. Subjects with heavy alcohol consumption showed markedly lower plasma immunoglobulin levels to PEth, potentially making them useful as a biomarker to distinguish heavy from moderate alcohol use.
Novel protein kinase C isoforms (PKC δ and ε) mediate early events in acute pancreatitis. Protein kinase D (PKD/PKD1) is a convergent point of PKC δ and ε in the signaling pathways triggered through CCK or cholinergic receptors and has been shown to activate the transcription factor NF-κB in acute pancreatitis. For the present study we hypothesized that a newly developed PKD/PKD1 inhibitor, CRT0066101, would prevent the initial events leading to pancreatitis. We pretreated isolated rat pancreatic acinar cells with CRT0066101 and a commercially available inhibitor Gö6976 (10 μM). This was followed by stimulation for 60 min with high concentrations of cholecystokinin (CCK, 0.1 μM), carbachol (CCh, 1 mM), or bombesin (10 μM) to induce initial events of pancreatitis. PKD/PKD1 phosphorylation and activity were measured as well as zymogen activation, amylase secretion, cell injury and NF-κB activation. CRT0066101 dose dependently inhibited secretagogue-induced PKD/PKD1 activation and autophosphorylation at Ser-916 with an IC(50) ∼3.75-5 μM but had no effect on PKC-dependent phosphorylation of the PKD/PKD1 activation loop (Ser-744/748). Furthermore, CRT0066101 reduced secretagogue-induced zymogen activation and amylase secretion. Gö6976 reduced zymogen activation but not amylase secretion. Neither inhibitor affected basal zymogen activation or secretion. CRT0066101 did not affect secretagogue-induced cell injury or changes in cell morphology, but it reduced NF-κB activation by 75% of maximal for CCK- and CCh-stimulated acinar cells. In conclusion, CRT0066101 is a potent and specific PKD family inhibitor. Furthermore, PKD/PKD1 is a potential mediator of zymogen activation, amylase secretion, and NF-κB activation induced by a range of secretagogues in pancreatic acinar cells.
Previous studies of pancreatic acinar cells characterized the effects of Ca(2+)-releasing secretagogues and substances, inducing acute pancreatitis on mitochondrial Ca(2+), transmembrane potential, and NAD(P)H, but dynamic measurements of the crucial intracellular adenosine triphosphate (ATP) levels have not been reported. Here we characterized the effects of these agents on ATP levels in the cytosol and mitochondria.
ATP levels were monitored using cytosolic- or mitochondrial-targeted luciferases.
Inhibition of oxidative phosphorylation produced a substantial decrease in cytosolic ATP comparable to that induced by inhibition of glycolysis. Cholecystokinin-8 (CCK) increased cytosolic ATP in spite of accelerating ATP consumption. Acetylcholine, caerulein, and bombesin had similar effect. A bile acid, taurolithocholic acid 3-sulfate (TLC-S); a fatty acid, palmitoleic acid (POA); and palmitoleic acid ethyl ester (POAEE) reduced cytosolic ATP. The ATP decrease in response to these substances was observed in cells with intact or inhibited oxidative phosphorylation. TLC-S, POA, and POAEE reduced mitochondrial ATP, whereas physiological CCK increased mitochondrial ATP. Supramaximal CCK produced a biphasic response composed of a small initial decline followed by a stronger increase.
Both glycolysis and oxidative phosphorylation make substantial contributions to ATP production in acinar cells. Ca(2+)-releasing secretagogues increased ATP level in the cytosol and mitochondria of intact isolated cells. TLC-S, POA, and POAEE reduced cytosolic and mitochondrial ATP. When cells rely on nonoxidative ATP production, secretagogues as well as TLC-S, POA, and POAEE all diminish cytosolic ATP levels.
The early pathogenetic steps that finally lead to acinar cell necrosis in acute pancreatitis have been characterized only scarcely as yet. Among a lot of hypotheses, one concept favors disturbances of cellular energy metabolism as a major factor that contributes to preterm cell decline. To investigate, whether an experimental acute pancreatitis alters cell respiration, the respiratory capacities of acinar cells isolated from rats with acute pancreatitis were measured. Acute pancreatitis was induced using Popper's model, i.e., a combination of duct obstruction, secretory stimulation, and mesenteric short-term ischemia with subsequent reperfusion. Acinar cells were isolated using a collagenase digestion technique. The respiratory rates of the isolated cells in suspension were measured at 37 degrees C in 100% oxygen-saturated N-(2-hydroxyethyl)piperazine-N'-2-ethanesulfonic acid-buffered Eagle's-minimal essential medium. Resting respiration of the acinar cells uniformly amounted to about 60 pmol of O2/s x 10(6) cells in both the control and the pancreatitis group. Cellular respiration could significantly be stimulated by stepwise uncoupling of oxidative phosphorylation by means of 2,4-dinitrophenol in all cell suspensions investigated. The maximum rate of stimulated respiration was diminished in the cells isolated from rats with acute pancreatitis as compared with the controls (79.3 +/- 5.0 vs. 160.2 +/- 15.5 pmol of O2/s x 10(6) cells, p < .05), however. This reduced respiratory load capacity of the acinar cells in acute pancreatitis reflects the restricted ability of the cells to increase respiration on enhanced cellular demand. Since mitochondrial respiration is coupled to oxidative phosphorylation, an altered energy-transforming potential of the acinar cells in acute pancreatitis becomes evident.(ABSTRACT TRUNCATED AT 250 WORDS)
Hypertriglyceridemia is an established cause of pancreatitis and has been suggested as a predisposing factor in alcohol and gallstone-induced pancreatitis. The aims of this study were to determine fasting and postprandial triglyceride levels of alcoholics with pancreatitis, alcoholics without pancreatitis, patients with previous gallstone pancreatitis, patients with choledocholithiasis, and healthy controls.
Oral lipid tolerance studies were performed in the above groups.
No relationship was found between alcoholic pancreatitis and hypertriglyceridemia, regardless of whether subjects were studied in the fasting state, after ingestion of fat, or after ingestion of fat with ethanol. Plasma triglyceride levels of alcoholics with pancreatitis remained similar to those of alcoholics without pancreatitis, but levels in both groups varied in relation to recent alcohol intake. Plasma triglyceride levels from both groups of alcoholics were greater than those of nonalcoholic healthy subjects. In addition, the previously reported association between postprandial hypertriglyceridemia and gallstone pancreatitis was not observed.
Plasma triglyceride levels do not account for individual susceptibility to either alcoholic or gallstone pancreatitis.
Suprastimulation of pancreatic acinar cells with specific agonists inhibits zymogen secretion and induces the formation of large basolateral blebs. Currently the molecular mechanisms that mediate this dramatic morphologic response are undefined. Further, it is unclear if blebbing represents a terminal or reversible event. Using computer-enhanced video microscopy of living acini we have found that these large blebs form rapidly (within 2-3 minutes) and exhibit ameboid undulations. They are induced by small increases in agonist concentration and require an energy-dependent phosphorylation event. Remarkably, the blebs are rapidly absorbed when agonist levels are reduced, indicating that blebbing is a reversible response to a physiological stimulus, not a terminal event. Morphological methods show that these dramatic changes in cell shape are accompanied by a marked reorganization of actin and myosin II at the basolateral domain. During 30 minutes of suprastimulation, both basolateral actin and myosin II gradually increase to form a ring centered at the necks of the blebs. Immunocytochemical and biochemical studies with a phospho-specific antibody to the myosin regulatory light chain reveal an activation of myosin II in suprastimulated acini that is completely absent in resting cells. Studies using cytoskeletal antagonistic drugs indicate that bleb formation and motility require actin remodeling concomitant with an activation of myosin II. This aberrant activation and reorganization of the actin-myosin cytoskeleton is likely to have detrimental effects on acinar cell function. Additionally, this mechanism of bleb formation may be conserved among other forms of physiological blebbing events.
Although alcoholism is a major cause of pancreatitis, the pathogenesis of this disorder remains obscure. Failure to produce experimental alcoholic pancreatitis suggests that ethanol may only increase predisposition to pancreatitis. This study sought to develop a model of ethanol pancreatitis by determining if an ethanol diet sensitizes rats to pancreatitis caused by cholecystokinin octapeptide (CCK-8).
Rats were fed intragastrically either control or ethanol diet for 2 or 6 weeks. The animals were then infused for 6 hours with either saline or CCK-8 at a dose of 3000 pmol. kg(-1). h(-1), which by itself did not induce pancreatitis. The following parameters were measured: serum amylase and lipase levels, pancreatic weight, inflammatory infiltration, number of apoptotic acinar cells, pancreatic messenger RNA (mRNA) expression of cytokines and chemokines, and nuclear factor (NF)-kappaB activity.
All measures of pancreatitis, as well as NF-kappaB activity and mRNA expression for tumor necrosis factor alpha, interleukin 6, monocyte chemotactic protein 1, macrophage inflammatory protein 2, and inducible nitric oxide synthase, were significantly increased only in rats treated with ethanol plus CCK-8.
An ethanol diet sensitizes rats to pancreatitis caused by CCK-8. The combined action of ethanol and CCK-8 results in NF-kappaB activation and up-regulation of proinflammatory cytokines and chemokines in the pancreas. These mechanisms may contribute to the development of alcoholic pancreatitis.
This article discusses zymogen activation within the acinar cell. The authors review advances in respect to the cellular mechanism involved in a premature intrapancreatic protease activation. Critical factors that determine the onset of premature protease activation appear to be the molecular structure of trypsinogen, the presence or absence of functionally intact lysosomal hydrolases, the pH in intracellular compartments, and the calcium signaling cascade in the pancreatic acinar cell.
Relationships between calcium signals and NADH responses were investigated in pancreatic acinar cells stimulated with calcium-releasing secretagogues. Cytosolic calcium signals were studied using Fura Red or calcium-sensitive Cl(-) current. Mitochondrial calcium was measured using Rhod-2. The highest levels of NADH autofluorescence were found around the secretory granule region. Stimulation of cells with physiological doses of cholecystokinin (CCK) triggered slow oscillations of NADH autofluorescence. NADH oscillations were clearly resolved in the mitochondrial clusters around secretory granules. Very fast apical calcium signals induced by acetylcholine (ACh) produced no detectable changes in NADH; slightly more extended apical (or preferentially apical) calcium transients triggered clear NADH responses. Triple combined recordings of cytosolic calcium, mitochondrial calcium and NADH revealed the sequence of development of individual signals: an increase in cytosolic calcium was accompanied by a slower mitochondrial calcium response followed by a delayed increase in NADH fluorescence. Recovery of cytosolic calcium was faster than recovery of mitochondrial calcium. NADH recovery occurred at elevated mitochondrial calcium levels. During the transient cytosolic calcium oscillations induced by intermediate doses of ACh, there was an initial increase in NADH fluorescence following the first calcium transient; each of the subsequent calcium responses produced biphasic NADH changes comprising an initial small decline followed by restoration to an elevated calcium level. During the higher-frequency sinusoidal calcium oscillations induced by higher doses of ACh, NADH responses fused into a smooth rise followed by a slow decline. Supramaximal doses of ACh and CCK produced single large NADH transients.
In the present study, we have investigated the effect of ethanol on amylase release in response to cholecystokinin octapeptide (CCK-8). We have also studied the effect of ethanol on cytosolic free Ca(2+) concentration ([Ca(2+)](c)) and reactive oxygen species (ROS) production by loading of cells with fura-2 and 5-(and-6)-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H(2)DCFDA), respectively. Our results show that stimulation of pancreatic acinar cells with CCK-8 induced a dose-dependent amylase secretion, resulting in a maximum at 0.3nM of 19.39+/-2.73% of the total content of amylase. Treatment of pancreatic acini with ethanol did not induce any significant effect on amylase release at a wide range of concentrations (1-50mM). In contrast, incubation of cells with 50mM ethanol clearly reduced amylase release stimulated by CCK-8. The inhibitory effect of ethanol on CCK-8-induced amylase secretion was abolished by dithiothreitol, a sulfhydryl reducing agent. Ethanol induced an increase in [Ca(2+)](c) resulting in a level higher than the prestimulation level both in the presence and in the absence of extracellular Ca(2+). Additionally, ethanol led to an increase in fluorescence of CM-H(2)DCFDA, reflecting an increase in oxidation. A decrease in oxidation was observed in the absence of extracellular Ca(2+) and in the presence of ethylene glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid. Similarly, when the cells were challenged in the presence of the intracellular Ca(2+) chelator 1,2-Bis(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and in the absence of extracellular Ca(2+), the responses to ethanol were reduced, although not completely inhibited. Taken together, our results suggest that ethanol induces generation of ROS by a Ca(2+)-dependent mechanism and reduces CCK-8-evoked amylase secretion in exocrine pancreatic cells.
The molecular mechanism of clinical alcohol-induced pancreatitis remains vague. We had reported that experimental high-dose cholecystokinin (CCK)-induced pancreatitis is in part because of excessive aberrant basolateral exocytosis. High-dose CCK caused Munc18c on basolateral plasma membrane (BPM) to dissociate from syntaxin (Syn)-4, activating Syn-4 to complex with plasma membrane (PM)-SNAP-23 and granule-VAMP to mediate basolateral exocytosis. We now hypothesize that alcohol could render the acinar cell BPM conducive to exocytosis by a similar mechanism. Weakly stimulating postprandial doses of alcohol (20-50 mM) inhibited postprandial low-dose CCK-stimulated secretion by blocking physiologic apical exocytosis and redirecting exocytosis to less-efficient basal PM (visualized by FM1-43 fluorescence imaging) and lateral PM sites (electron microscopy). Alcohol or low-dose CCK had no effect on PM-Munc18c, but alcohol preincubation enabled low-dose CCK to displace Munc18c from BPM, leading to SNARE complex assembly in the BPM. Similarly, alcohol diet-fed rats did not exhibit morphologic defects in the pancreas nor affected PM-Munc18c behavior, but subsequent intraperitoneal injections of low-dose CCK analog cerulein caused Munc18c displacement from BPM and cytosolic degradation, which contributed to pancreatitis. We conclude that alcohol induces BPM-Munc18c to become receptive to postprandial CCK-induced displacement into the cytosol, a process which facilitates SNARE complex assembly that in turn activates restricted BPM sites to become available for aberrant exocytosis into the interstitial space, where zymogen activation would take place and cause pancreatitis.
In this study, we have examined the effects of rotenone in primary cultures of hippocampal and dopaminergic neurons in order to obtain insights into the possible mechanisms underlying the neurotoxic effects of this pesticide. The results obtained indicate that a 48-h exposure to rotenone (0.1 microM) produces a complete and selective suppression of axon formation. This effect was dose dependent, not accompanied by changes in microtubule organization, and reversible after washout of the agrochemical from the tissue culture medium. Interestingly, pull-down assays revealed that rotenone decreases Cdc42 and Rac activities, whereas increasing that of Rho. In accordance with this, treatment of neuronal cultures with cytochalasin D, an actin-depolymerizing drug, or with the Rho-kinase inhibitor Y27632, or overexpression of Tiam1, a guanosine nucleotide exchange factor for Rac, reverts the inhibitory effect of rotenone on axon formation. Taken together, our data suggest that at least some of the neurotoxic effects of rotenone are associated with an inhibition of actin dynamics through modifications of Rho-GTPase activity.