Fatty Acid Ethyl Ester Synthase Activity Research Articles

Alcohol oxidizing enzymes and ethanol-induced cytotoxicity in rat pancreatic acinar AR42J cells

Alcoholic chronic pancreatitis (ACP) is a serious inflammatory disease causing significant morbidity and mortality. Due to lack of a suitable animal model, the underlying mechanism of ACP is poorly understood. Chronic alcohol abuse inhibits alcohol dehydrogenase (ADH) and facilitates nonoxidative metabolism of ethanol to fatty acid ethyl esters (FAEEs) in the pancreas frequently damaged during chronic ethanol abuse. Earlier, we reported a concentration-dependent formation of FAEEs and cytotoxicity in ethanol-treated rat pancreatic tumor (AR42J) cells, which express high FAEE synthase activity as compared to ADH and cytochrome P450 2E1. Therefore, the present study was undertaken to investigate the role of various ethanol oxidizing enzymes in ethanol-induced pancreatic acinar cell injury. Confluent AR42J cells were pre-treated with inhibitors of ADH class I and II [4-methylpyrazole (MP)] or class I, II, and III [1,10-phenanthroline (PT)], cytochrome P450 2E1 (trans-1,2-dichloroethylene) or catalase (sodium azide) followed by incubation with 800 mg% ethanol at 37°C for 6 h. Ethanol metabolism, cell viability, cytotoxicity (apoptosis and necrosis), cell proliferation status, and formation of FAEEs in AR42J cells were measured. The cell viability and cell proliferation rate were significantly reduced in cells pretreated with 1,10-PT + ethanol followed by those with 4-MP + ethanol. In situ formation of FAEEs was twofold greater in cells incubated with 1,10-PT + ethanol and ∼1.5-fold in those treated with 4-MP + ethanol vs. respective controls. However, cells treated with inhibitors of cytochrome P450 2E1 or catalase in combination of ethanol showed no significant changes either for FAEE formation, cell death or proliferation rate. Therefore, an impaired ADH class I-III catalyzed oxidation of ethanol appears to be a key contributing factor in ethanol-induced pancreatic injury via formation of nonoxidative metabolites of ethanol.

Alcohol linked to enhanced angiogenesis in rat model of choroidal neovascularization

One of the pathologic complications of exudative (i.e. wet-type) age-related macular degeneration (AMD) is choroidal neovascularization (CNV). The aim of this study was to investigate whether chronic and heavy alcohol consumption influenced the development of CNV in a rat model. The oxidative metabolism of alcohol is minimal or absent in the eye, so that ethanol is metabolized via a nonoxidative pathway to form fatty acid ethyl esters (FAEE). Fatty acid ethyl ester synthase (FAEES) was purified from the choroid of Brown Norway (BN) rats. The purified protein was 60 kDa in size and the antibody raised against this protein showed a single band on western blot. BN rats on a regular diet were fed alcohol for 10 weeks. Control rats were fed water with a regular diet and pair-fed control rats were fed regular diet, water and glucose. We found that FAEES activity was increased 4.0-fold in the choroid of alcohol-treated rats compared with controls. The amount of ethyl esters produced in the choroid of 10 week alcohol-fed rats was 7.4-fold more than rats fed alcohol for 1 week. The increased accumulation of ethyl esters was associated with a 3.0-fold increased expression of cyclin E and cyclin E/CDK2; however, the level of the cyclin kinase inhibitor, p27Kip, did not change. The increased accumulation of ethyl esters was also associated with 3.0-fold decreased expression of APN in the choroid. We also found that the size of CNV increased by 28% in alcohol-fed rats. Thus, our study showed that chronic, heavy alcohol intake was associated with both an increased accumulation of ethyl esters in the choroid and an exacerbation of the CNV induced by laser treatment. These results may provide insight into the link between heavy alcohol consumption and exudative AMD.

Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri- o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-( o-cresyl)-4 H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di- o-tolyl- o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase–esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase–esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase–esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase–esterase activities.

Mechanism of differential inhibition of hepatic and pancreatic fatty acid ethyl ester synthase by inhibitors of serine-esterases: in vitro and cell culture studies

Earlier, we have shown that rat hepatic and pancreatic fatty acid ethyl ester (FAEE) synthases are structurally and functionally similar to rat liver carboxylesterase (CE) and pancreatic cholesterol esterase (ChE), respectively. We have also reported that only hepatic FAEE synthase is inhibited by tri-o-tolylphosphate (TOTP) in vivo and in human hepatocellular carcinoma (HepG2) cells. The metabolism of TOTP is a prerequisite for the inhibition of hepatic FAEE synthase as well as esterase activity. To further elucidate the mechanism of such differential inhibition by inhibitors of serine esterases, we synthesized two metabolites of TOTP, 2-(o-cresyl)-4H-1:3:2-benzodioxaphosphoran-2-one (CBDP; cyclic saligenin phosphate) and di-o-tolyl-o-(∝-hydroxy)tolylphosphate (HO-TOTP), and one ChE inhibitor, 3-benzyl-6-chloro-2-pyrone (3-BCP). The inhibitory effect of CBDP, HO-TOTP, and 3-BCP on FAEE synthase and esterase activity was studied using rat hepatic and pancreatic postnuclear (PN) fractions, commercial porcine hepatic CE and pancreatic ChE, and in HepG2 and rat pancreatic tumor (AR42J) cell lines. Only HO-TOTP and CBDP inhibited FAEE synthase as well as esterase activity of hepatic PN fraction and commercial CE and ChE in a concentration-dependent manner, and the inhibition was found to be irreversible. However, no inhibition was found in pancreatic PN fraction by both TOTP metabolites and 3-BCP. Although 3-BCP inhibited only the esterase activity of commercial ChE in a concentration-dependent manner, the activity was reversible within 30 min of incubation. Studies with HepG2 cells also showed a significant inhibition of FAEE synthase–esterase activity by CBDP and HO-TOTP within 15 min of incubation, while no inhibition was observed in AR42J cells. 3-BCP did not inhibit FAEE synthase–esterase activity either in HepG2 or AR42J cells. Such differential inhibitory effect of the TOTP metabolites on hepatic and pancreatic FAEE synthase–esterase is supported by our earlier in vivo and in vitro studies. Further investigations are needed to understand the biochemical mechanism(s) of inactivation of TOTP metabolites and 3-BCP in the pancreas and AR42J cells towards FAEE synthase–esterase activities.

Modulation of Fatty Acid Methyl Esters in Rats Pretreated with Tri-o-Tolyl Phosphate

Formation and toxicity of fatty acid methyl esters (FAMEs) have been reported both in vitro and in vivo. In previous studies, it was shown that fatty acid ethyl ester synthase (FAEES), which catalyzes the formation of FAMEs, also expresses esterase activity. Therefore, it was hypothesized that inhibitors of esterases such as tri-o-tolyl phosphate (TOTP) can modulate the formation of FAMEs. To test this, four groups of rats were used. Group 1 served as control (vehicle only). Group 2 was treated with methanol only (3 g/kg via gavage), group 3 was given TOTP only (100 mg/kg ip in corn oil), and group 4 was administered TOTP as in group 3, followed by methanol after 18 h. Three hours after exposure, animals were sacrificed and FAEES activity and FAME levels were measured in blood, liver, pancreas, and brown fat. About 95% of FAEES activity was inhibited in the liver and whole blood of TOTP-treated rats (group 3) but no inhibition was observed in the pancreas or brown fat. Total hepatic FAMEs were found to be lowest for the TOTP-treated group (3) and highest in the methanol-treated animals (group 2). Total pancreatic FAMEs in different groups were not statistically different, while significant increases were observed in the brown fat in both methanol-treated groups. To verify that the oxidative metabolism of methanol was unaffected by TOTP, alcohol dehydrogenase activity was also measured and found to be unchanged in any group as compared to control. These results demonstrate that the formation of FAMEs can be modulated in the liver and probably in blood, but not in the pancreas or brown fat by the inhibitors of FAEES.

Ethanol metabolism and transcription factor activation in pancreatic acinar cells in rats

Background & Aims:Ethanol metabolism by pancreatic acinar cells and the role of its metabolites in ethanol toxicity to the pancreas remain largely unknown. Here, we characterize ethanol metabolism in pancreatic acinar cells and determine the effects of ethanol metabolites on nuclear factor κB (NF-κB) and activator protein (AP)-1, transcription factors that are activated in pancreatitis and mediate expression of inflammatory molecules critical for this disease. Methods:We measured activities of fatty acid ethyl ester (FAEE) synthase and alcohol dehydrogenase (ADH), as well as accumulation of ethanol metabolites. We measured the effects of ethanol and its metabolites on NF-κB and AP-1 activation by using a gel shift assay. Results: Pancreas metabolizes ethanol via both oxidative and nonoxidative pathways. Acinar cells are the main source of ethanol metabolism in the pancreas. Compared with the liver, FAEE synthase activity in the pancreas is greater, whereas that of ADH is much less. FAEEs activated NF-κB and AP-1, whereas acetaldehyde inhibited NF-κB activation. Ethanol decreased NF-κB binding activity in acinar cells, which was potentiated by cyanamide. Conclusion:Oxidative and nonoxidative ethanol metabolites regulate transcription factors differently in pancreatic acinar cells. Ethanol may regulate NF-κB and AP-1 positively or negatively, depending on which metabolic pathway's effect predominates. These regulatory mechanisms may play a role in ethanol toxicity to the pancreas.

Linkage of oxidative and nonoxidative ethanol metabolismin the pancreas and toxicity of nonoxidative ethanol metabolites for pancreatic acinar cells

Background. Alcohol abuse is a major cause of pancreatic damage. Recent experimental evidence suggests that fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites, injure pancreatic acinar cells. Linkage between oxidative and nonoxidative metabolism of ethanol in the pancreas may contribute to increased FAEE levels. Methods. To study the association between oxidative and nonoxidative ethanol metabolism, FAEE concentration and FAEE synthase activity in rat pancreatic and liver homogenates incubated with ethanol were evaluated with and without inhibitors of oxidative ethanol metabolism. For toxicity studies, trypsinogen activation peptide synthesis as a measure of pancreatic cell injury was quantitated in unstimulated and cerulein-stimulated isolated pancreatic acinar cells incubated with ethanol or FAEE. Results. Inhibition of oxidative ethanol metabolism results in a 2- to 3-fold increase in nonoxidative ethanol metabolism to FAEE in pancreas and in liver. Both ethanol and FAEE induce increased intracellular trypsinogen activation by more than 50% in the presence of physiologic concentrations of cerulein in vitro. Conclusions. These findings demonstrate that the inhibition of oxidative ethanol metabolism results in an increase in flux through the nonoxidative pathway and support the proposition that alcohol-induced pancreatic injury is mediated at least in part by FAEE, which are important products of pancreatic ethanol metabolism.(Surgery 2001;129:736-44.)

Purification and characterization of rat hepatic microsomal low molecular weight fatty acid ethyl ester synthase and its relationship to carboxylesterases.

We reported purification of a high molecular weight (HMW) (ca. 180 kD) and a low molecular weight (LMW) (ca. 60 kD) protein fractions from digitonized rat liver microsomes using ammonium sulfate precipitation followed by ion exchange and gel filtration column chromatography. Both fractions expressed fatty acid ethyl ester (FAEE) synthase as well as p-nitrophenyl acetate (PNPA)-hydrolyzing (esterase) activities. The HMW fraction was found to be a trimer with subunit molecular weight ca. 60 kD and structurally and functionally similar to rat hepatic microsomal carboxylesterase (CE, pI 6.1) and adipose tissue FAEE synthase. In this article, we report further purification and characterization of the LMW (minor) fraction expressing FAEE synthase activity and its structural and functional relationship to hepatic microsomal CEs. Using isoelectric focusing (IEF) followed by gel filtration-high-performance liquid chromatography (GF-HPLC), five proteins were purified, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. The isoelectric point values of 6.5, 5.8, 5.6, 5.3, and 5.0 were found for the purified LMW proteins by IEF and each showed a peak corresponding to ca. 60 kD molecular weight by GF-HPLC, which expressed FAEE synthase as well as PNPA-hydrolyzing activity. Sodium dodecyl sulfate-polyacrylamide gel elecrophoresis (SDS-PAGE) analysis of the GF-HPLC purified LMW proteins revealed that these proteins are monomers (ca. 60 kD). All the purified LMW proteins cross-reacted with antibodies to rat adipose tissue FAEE synthase. Coelution of PNPA-hydrolyzing and FAEE synthase activity at each step of purification and cross-reactivity with rat adipose tissue FAEE synthase antibodies suggest that the purified proteins are related to various hepatic microsomal CEs. This conclusion is further supported by the homology of N-terminal amino acid sequence of the purified LMW proteins to various hepatic microsomal CEs and protease precursors. Therefore, LMW FAEE synthase activity most probably is expressed by various isozymes of hepatic microsomal CEs, which are also involved in the biotransformation of xenobiotic alcohols and amines.

Effects of acetyl-L-carnitine on the formation of fatty acid ethyl esters in brain and peripheral organs after short-term ethanol administration in rat.

Increasing evidence suggests that Fatty acid ethyl esters (FAEE) play a central role in ethanol induced organ damage. In the current study we measured FAEE formation in rats after short-term oral administration of ethanol, in the presence and absence of pre-treatment with acetyl-L-carnitine. Ethanol treatment caused a significant increase in the levels of FAEE, particularly in the brain and heart, but also in the kidney and liver. Increases in FAEE were associated with a significant increase in FAEE synthase activity, GSH transferase activity, and lipid hydroperoxide levels. Pretreatment with acetyl-L-carnitine resulted in a significant reduction of FAEE accumulation, decrease in FAEE synthase and GSH transferase activities, and lipid hydroperoxide levels. Administration of acetyl-L-carnitine greatly reduced the metabolic abnormalities due to non-oxidative ethanol metabolism, through an increment in lipid metabolism/turnover and by the modulation of the activities of enzymes associated with FAEE synthesis. These results suggest a potentially important pharmacological role for acetyl-L-carnitine in the prevention of alcohol-induced cellular damage.

Fatty Acid Ethyl Ester Synthesis in the Preparation of Scotch Whiskey

Fatty acid ethyl esters (FAEE), nonoxidative ethanol metabolites present in human organs commonly damaged by ethanol abuse, have been implicated as mediators of organ damage. FAEE are additives in various foods and beverages to provide flavor or fragrance, and therefore are common dietary lipid constituents. We hypothesized that FAEE could be generated during alcoholic beverage production because fatty acids are present within microorganisms and ethanol is generated during the fermentation process. In this report, we demonstrate that FAEE are present in commercially available scotch beverages, and that in the preparation of scotch, FAEE can be produced during the fermentation reaction as a result of FAEE synthase activity in the yeast. Following ingestion of scotch, preformed FAEE are delivered to GI tract. The consequences of ingestion of FAEE in scotch, if any, remain to be determined.

Formation of fatty acid ethyl esters in rat liver microsomes. Evidence for a key role for acyl-CoA: ethanol O-acyltransferase.

Fatty acid ethyl esters have been detected in high concentrations in organs commonly damaged by alcohol abuse and are regarded as being important non-oxidative metabolites of ethanol. The formation of fatty acid ethyl esters (FAEEs) has been ascribed to two enzymic activities, acyl-CoA : ethanol O-acyltransferase (AEAT) and FAEE synthase. In the present study we determined AEAT and FAEE synthase activities in isolated rat liver microsomes and further characterized the microsomal AEAT activity in more detail. The determined AEAT and FAEE synthase activities were found to be similar (about 1.7 nmol.min-1.mg-1). However, the AEAT activity was increased about sixfold by the addition of 250 microm bis-(4-nitrophenyl) phosphate (a serine esterase inhibitor) to the incubation whereas FAEE synthase activity was completely inhibited. p-Hydroxymercuribenzoic acid (a cysteine-reacting compound) also stimulated AEAT activity (about fourfold) but had no effect on FAEE synthase activity. The effects of the inhibitors suggest that the formation of FAEEs by AEAT was severely counteracted by enzymic hydrolysis of the substrate (acyl-CoA) and to a lesser extent the product by serine esterases. dl-Melinamide, a hypocholesterolaemic drug, was found to be a very potent inhibitor of AEAT activity with an IC50 value of about 2.5 microm. Furthermore, we compared the activities of two purified microsomal carboxylesterases, ES-4 and ES-10, and identified ES-4 as the enzyme responsible for hydrolysis of FAEEs. The two carboxyesterases were also tested for FAEE synthase activity, but neither had any detectable activity. Esterase ES-4 was found to have some AEAT activity, but it was low. When measured under optimal conditions without competing hydrolysis the capacity of AEAT is thus considerably higher than FAEE synthase and the results are consistent with an important role for AEAT in the formation of ethyl esters. As the ratio acyl-CoA/non-esterified fatty acids is high under normal conditions, AEAT is probably the most important enzyme in fatty acid ethyl ester formation.

Effects of acute and chronic ethanol exposure on fatty acid ethyl ester synthases in mouse cerebellar membranes.

Fatty acid ethyl esters (FAEE), the products of esterification of fatty acids with ethanol (EtOH), are shown to cause organ injury in chronic alcohol abusers. Their formation is catalysed by the enzyme FAEE synthase which is present in both animal and human brain. In the present study, we investigated the effects of acute and chronic EtOH exposure on FAEE synthase activity in crude cerebellar membrane preparation of mice, using oleic, linoleic and arachidonic acids as substrates. The results indicate that FAEE synthase activity exists in synaptosomal membranes and the activity of this enzyme varied with various fatty acid substrates. The synthase activity was optimal in the membranes from the animals exposed acutely to EtOH when oleic acid was used as a substrate. A 14% increase in the incorporation of oleic acid was observed in the membranes from animals exposed to acute dose of EtOH. However, there was a 10% reduction in the synthase activity when arachidonic acid was used as a substrate in the membrane preparations from mice exposed chronically to EtOH. The results suggest that substrate specificity for FAEE synthase varied with the duration of exposure to EtOH.

Reduced Fatty Acid Ethyl Ester Synthase Activity in the White Blood Cells of Alcoholics

Fatty acid ethyl esters (FAEEs), esterification products of ethanol and fatty acids, have been implicated as mediators of ethanol-induced organ damage. It has been shown that FAEE synthase, the enzyme responsible for the formation of FAEE, is present selectively in the organs commonly damaged by ethanol abuse. Recently, we have made the observation that FAEEs are also present in the serum after ethanol ingestion. The current study was performed to determine whether cellular elements of the blood and/or plasma are capable of synthesizing FAEEs from fatty acids and ethanol. Heparinized blood samples were collected from 10 healthy volunteers, and the red blood cells, platelets, plasma, and several white blood cell populations were assayed for FAEE synthase activity. Blood samples from control subjects and individuals admitted to an alcoholic detoxification unit at a local hospital were also assayed for FAEE synthase activity. We observed that the FAEE synthase activity is present in whole blood, primarily within white blood cells. Fractionation of the white blood cells revealed that the lymphocyte-monocyte fraction isolated using Ficoll-hypaque contained approximately 3.5-fold higher activity than the granulocyte fraction. The cell type that contained the highest FAEE synthase activity (1220 pmol/hr/10(6) cells) was the natural killer (NK) cell population. B cells contained approximately 40% of the enzyme activity found in NK cells, and the B-cell activity was slightly greater than that found in CD4+ and CD8+ T cells. Having shown that FAEE synthase exists in a blood cell, we subsequently demonstrated that alcoholic individuals have approximately half the white blood cell FAEE synthase activity of that found in normal controls. We also demonstrated that white blood cell FAEE synthase could be induced nearly 2-fold upon ingestion of 2 oz of scotch whiskey for 6 days. The enzyme activity returned to baseline levels despite ingestion of 2 oz of scotch whiskey/day for 3 additional days. These data indicate that ethanol ingestion results in increased FAEE production, particularly by NK cells. FAEE synthesis after ethanol ingestion may explain the presence of FAEE in the serum. The lower enzyme activity observed in white blood cells of alcoholics from a detoxification center may be the result of years of ethanol abuse or it may be that alcoholics congenitally have low levels of FAEE synthase. If the latter is true, this finding may explain in part the genetic predisposition of many alcoholic individuals to ethanol abuse.

Comparison of the induction of rat glutathione S-transferase and fatty acid ethyl ester synthase activities

Fatty acid ethyl esters (FAEE) are formed following the administration of ethanol and have previously been associated with lexicological effects in animals and humans. It has been suggested that the enzyme responsible, FAEE synthase, has both structural and catalytic properties very similar to a glutathione S-transferase (GST). Since GSTs are inducible, their induction could be associated with enhanced FAEE formation and toxicity. In the present study, rats were administered β-naphthoflavone, phenobarbital, ethanol, or Aroclor 1254, and hepatic FAEE synthase and GST activities were measured. β-Naphthoflavone and ethanol did not induce either activity. Phenobarbital increased GST activity in the liver but not in lung or pancreas. Only Aroclor 1254, which increased GST activity in liver and pancreas, increased FAEE synthase activity and then only in the liver. Thus, in comparison with GST activity, FAEE synthase activity is very limited in its ability to be induced.