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

Abstract

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.

Keywords: ACUTE Pancreatitis; Alcohol-Induced Injury; Calcium; Ethanol; Pancreatic Damage.

Figure 1

Effects of ethanol (EtOH), palmitoleic acid (POA), 4-methylpyrazole (4-MP) and 3-benzyl-6-chloro-2-pyrone (3-BCP) on [Ca²⁺]_C in pancreatic acinar cells. (A) Typical fluorescence images showing changes of [Ca²⁺]_C (Fluo4, green) before (t=120 s) and after (t=800 s) application of combinations of ethanol (10 mmol/L), POA (20 μmol/L), 4-MP (100 μmol/L) and 3-BCP (10 μmol/L). Addition of ethanol/POA/4-MP caused sustained elevation of [Ca²⁺]_c seen at 800 s. (B) Combination of ethanol/POA (wine) induced predominantly oscillatory increases of [Ca²⁺]_C, while additional presence of 4-MP (100 μmol/L) promoted a shift towards sustained increases (red); 4-MP alone was without effect (blue). The addition of 3-BCP abolished sustained rises induced by ethanol/POA/4-MP (cyan). Cumulative data expressed as percentage of cells exhibiting sustained rises (inset) (F/F₀=>1.5) at 800 s (total n=159 cells).

Figure 2

Effects of ethanol (EtOH), palmitoleic acid (POA), 4-methylpyrazole (4-MP) and 3-benzyl-6-chloro-2-pyrone (3-BCP) on mitochondrial membrane potential (Δψ_M) in pancreatic acinar cells. (A) Typical fluorescence (glow) images showing progressive loss (six images, left) or maintenance (two images, right) of Δψ_M to ethanol/POA/4-MP combination in absence (left), or presence (right) of 3-BCP. Complete mitochondrial depolarisation induced by carbonyl cyanide 3-chlorophenylhydrazone (CCCP;10 μmol/L) is seen at 900 s. (B) Graph showing ethanol/POA-induced loss of Δψ_M (n=39), exacerbated by 4-MP (n=44). 4-MP alone was without effect (n=23). 3-BCP abolished effects of ethanol/POA/4-MP (n=36). Summarised data (inset) show mean % depolarisation±SE for each application. (C) Progressive loss of mitochondrial reduced nicotinamide adenine dinucleotide (phosphate) (NAD(P)H) fluorescence induced by ethanol/POA/4-MP (red, n=44) was abolished by 3-BCP (cyan, n=36). Summarised data (inset) show changes of NAD(P)H fluorescence, expressed as mean±SE in mitochondria-specific (mito) versus mitochondria-free (cytosolic; cyto) regions, at baseline (70–100 s) and during stimulation (770–800 s) (* and ^# p<0.05 compared to control).

Figure 3

Mitochondrial localisation and activation of fatty acid ethyl ester probe (palmitoleic acid (POA)-Fluor) in pancreatic acinar cells. (A) Light-transmitted and fluorescent images of acinar cell doublet (inset) at time of membrane rupture (breakthrough) show localised fluorescence at patch-pipette tip as POA-Fluor activation by hydrolases released fluorescein. Time-dependent fluorescence rise (green) was associated with inward Ca²⁺-activated Cl⁻ currents (ICl_Ca), whereas no fluorescence was detected in the adjacent cell. (B) Light-transmitted and fluorescence images, and (C) subcellular regions of interest show predominantly mitochondrial distribution of fluorescence (fluorescein:NAD(P)H co-localisation), consistent with mitochondrial probe activation. [Ca²⁺]_C levels increased over time (sustained inward ICl_Ca with superimposed transients), accompanied by a concomitant decrease of NAD(P)H (not seen in non-patched, adjacent cell), consistent with fatty acid-induced mitochondrial inhibition.

Figure 4

Importance of non-oxidative metabolism of ethanol and oxidative metabolism of ethanol in pancreatic acinar cell death. (A) Levels of apoptosis (caspase; green) and necrosis (PI; red) were increased by palmitoleic acid ethyl ester (POAEE;50–200 μmol/L) compared with controls, whereas acetaldehyde (ACETAL, 100–200 μmol/L) was without effect. (B) Ethanol (EtOH; 10 mmol/L), palmitoleic acid (POA;20 μmol/L) or 4-methylpyrazole (4-MP;100 μmol/L), applied alone, did not increase caspase activation (green) or PI uptake (red). However, a combination significantly increased apoptotic and necrotic cell death pathway activation. (C) Inhibition of carboxylester lipase with 3-benzyl-6-chloro-2-pyrone (3-BCP;10 μmol/L) reversed ethanol/POA-induced cell death precipitated by 4-MP (expressed as % total cells; mean±SE; numbers in parentheses indicate cells assessed for each experimental condition, *p<0.05 compared to control values).

Figure 5

Localisation of carboxylester lipase (CEL) in pancreatic acinar cells and lobules. (A) Light-transmitted and fluorescent images of acinar cells showing CEL (green) location in the apical granular region, surrounded by peri-granular mitochondria (mitochondrial peptidyl-prolyl cis-trans isomerase: red). Nuclei co-stained with Hoechst 33342 (blue). (B) Similar distribution of CEL in unstimulated intact tissue. Treatment with physiological cholecystokinin (10 pM for 30 min) caused CEL to redistribute into the lumen between acinar clusters suggestive of secretion. (C) A diffuse intragranular and extragranular (apical and basolateral) distribution of CEL was observed in intact pancreatic lobules obtained from mice treated with ethanol/POA combination to induce alcoholic acute pancreatitis (i.p. ethanol (1.35 g/kg) and POA (150 mg/kg)).

Figure 6

Proposed mechanism of fatty acid ethyl ester (FAEE) generation by carboxylester lipase (CEL) and inhibition by 3-benzyl-6-chloro-2-pyrone (3-BCP). (A) (i) Molecular docking interactions predict binding of palmitoleic acid (POA) in the active site of CEL via interaction with Ser194 and His435 residues suggesting feasibility of mechanism-based esterification. (ii) 2D representation of the binding mode of POA in the active site indicates that the Ser194 hydroxyl group can react with the POA carbonyl group to afford a POA-Ser complex; ethanol easily displaces Ser from the complex forming POA ethyl ester (POAEE). (iii) Mechanism-based esterification of POA to POAEE. (B) (i) Binding of 3-BCP in CEL active site indicates interaction with Ser194 (via hydrogen bonding), further stabilised by two π-interactions with Phe324 and Trp227. (ii) Mechanism-based reaction of 3-BCP with Ser194 residue resulting in formation of 3-BCP-enzyme complex which is inhibitory for FAEE formation.

Figure 7

Features of fatty acid ethyl ester-induced acute pancreatitis induced by palmitoleic acid (POA)/ethanol. Mice received two intraperitoneal injections of ethanol (1.35 g/kg) in combination with POA at 0, 10, 20, 80 and 150 mg/kg, and mice were sacrificed 24 h after first injection. (A) Representative H&E images of histology slides from pancreas of mice treated with ethanol with POA. (B) Ethanol with POA (150 mg/kg) caused alveolar membrane thickening and inflammatory infiltration of the lung but did not induce any significant damage to liver, kidney or heart. Effects of ethanol with POA on (C) serum amylase, (D) Pancreatic trypsin activity and (E) Pancreatic myeloperoxidase activity (normalised). *p<0.05 compared to POA alone group. Values are mean±SE of 4–6 mice. Magnification: ×200.

Figure 8

Protective effects of 3-benzyl-6-chloro-2-pyrone (3-BCP) on biomarkers of fatty acid ethyl ester-induced acute pancreatitis (FAEE-AP) induced by palmitoleic acid (POA)/ethanol. FAEE-AP was induced by two intraperitoneal injections of ethanol (1.35 g/kg) and POA (150 mg/kg). The effects of inhibition of oxidative metabolism of ethanol (OME) with 4-methylpyrazole (4-MP; 10 mg/kg) or non-OME with 3-BCP (30 mg/kg), given on the first injection of POA/ethanol, were assessed on (A) Serum amylase, (B) Pancreatic trypsin activity, (C) Pancreatic myeloperoxidase activity (normalised) and (D) Serum interleukin-6 (IL-6). Control mice received saline injections only. Mice were sacrificed 24 h after the first injection. *p<0.05 compared to both saline and POA controls, ^#p<0.05 compared to the POA/ethanol group. Values are mean±SE of 6 mice.

Figure 9

Protective effects 3-benzyl-6-chloro-2-pyrone (3-BCP) on histological parameters of alcoholic acute pancreatitis. (A) Representative H&E images of pancreas histology slides from treatment groups; combinations of ethanol (1.35 g/kg), palmitoleic acid (POA; 150 mg/kg), 4-methylpyrazole (4-MP; 10 mg/kg) and 3-BCP (30 mg/kg). (B)(i) Overall histopathological score and breakdown components: (ii) oedema, (iii) inflammation and (iv) necrosis. All detrimental changes induced by ethanol/POA/4-MP were significantly ameliorated by 3-BCP. (*p<0.05 compared to both saline and POA controls, ^#p<0.05 compared to ethanol/POA group. Values are mean±SE of 6 mice. Magnification: ×200).