ATP-independent fatty acyl-coenzyme A synthesis from phospholipid: coenzyme A-dependent transacylation activity toward lysophosphatidic acid catalyzed by acyl-coenzyme A:lysophosphatidic acid acyltransferase

CoA-dependent transacylation activity in microsomes is known to catalyze the transfer of fatty acids between phospholipids and lysophospholipids in the presence of CoA without the generation of free fatty acids. We previously found a novel acyl-CoA synthetic pathway, ATP-independent acyl-CoA synthesis from phospholipids. We proposed that: 1) the ATP-independent acyl-CoA synthesis is due to the reverse reaction of acyl-CoA:lysophospholipid acyltransferases and 2) the reverse and forward reactions of acyltransferases can combine to form a CoA-dependent transacylation system. To test these proposals, we examined whether or not recombinant mouse acyl-CoA:1-acyl-sn-glycero-3-phosphate (lysophosphatidic acid, LPA) acyltransferase (LPAAT) could catalyze ATP-independent acyl-CoA synthetic activity and CoA-dependent transacylation activity. ATP-independent acyl-CoA synthesis was indeed found in the membrane fraction from Escherichia coli cells expressing mouse LPAAT, whereas negligible activity was observed in mock-transfected cells. Phosphatidic acid (PA), but not free fatty acids, served as an acyl donor for the reaction, and LPA was formed from PA in a CoA-dependent manner during acyl-CoA synthesis. These results indicate that the ATP-independent acyl-CoA synthesis was due to the reverse reaction of LPAAT. In addition, bacterial membranes containing LPAAT catalyzed CoA-dependent acylation of LPA; PA but not free fatty acid served as an acyl donor. These results indicate that the CoA-dependent transacylation of LPA consists of 1) acyl-CoA synthesis from PA through the reverse action of LPAAT and 2) the transfer of the fatty acyl moiety of the newly formed acyl-CoA to LPA through the forward reaction of LPAAT.     

A rapid assay of acyl-coenzyme A:lysolecithin acyltransferase activity

A simple and rapid procedure for the assay of acyl-coenzyme A:1-acyl-sn-glycero-3-phosphocholine acyltransferase (lysolecithin acyltransferase, LLAT [EC 2.3.1.23]) activity in crude enzyme preparations is described. The incubation system utilizes lysolecithin and [1-14C]-oleoyl-coenzyme A as substrates. Labeled fatty acid released due to accompanying acyl-coenzyme A hydrolase [EC 3.1.2.2]activity is first removed by di-isopropyl ether extraction. The labeled lecithin produced due to LLAT action is then quantitatively recovered by partition of the incubation medium with di-isopropyl ether-n-butanol 60:40 (v/v). Selective extraction of the labeled lecithin formed and avoidance of customary thin-layer chromatographic isolation procedures permits assay of LLAT activity with excellent accuracy at a substantial saving of time. The entire assay can be completed in less than 30 min as compared to 2-3 hrs when following conventional procedures.     

Eicosapentaenoic acid inhibits synthesis and secretion of triacylglycerols by cultured rat hepatocytes

Primary cultures of rat hepatocytes were used to study the effects of eicosapentaenoic and oleic acid on synthesis and secretion of triacylglycerols associated with very low density lipoproteins. From the experiments the following was observed. Oleic acid markedly stimulates secretion as well as synthesis of triacylglycerols, whereas eicosapentaenoic acid causes very little or no increase in secretion or synthesis as compared to a fatty-acid-free medium. The effects could already be observed after 15 min incubation. The inhibitory effect of eicosapentaenoic acid is reversible within 1-2 h. Eicosapentaenoic acid inhibits much of the stimulatory effect of oleic acid on synthesis and secretion of triacylglycerols. The cellular uptake of eicosapentaenoic acid is somewhat higher than that of oleic acid and the metabolism of these fatty acids to acid-soluble materials is similar. Eicosapentaenoic acid does not affect the secretory pathway of triacylglycerols per se. From these results it may be concluded that the mechanism for the inhibitory effect of eicosapentaenoic acid on triacylglycerol secretion is probably via reduced triacylglycerol synthesis.     

Regulation of hepatic cholesterol ester hydrolase and acyl-coenzyme A:cholesterol acyltransferase in the rat

Cholesterol exists within the hepatocyte as free cholesterol and cholesteryl ester. The proportion of intrahepatic cholesterol in the free or ester forms is governed in part by the rate of cholesteryl ester formation by acyl-coenzyme A:cholesterol acyltransferase (ACAT) and cholesteryl ester hydrolysis by neutral cholesterol ester (CE) hydrolase. In other cell types both ACAT and CE hydrolase activities are regulated in response to changes in the need for cellular free cholesterol. In rats, we performed a variety of experimental manipulations in order to vary the need for hepatic free cholesterol and to examine what effect, if any, this had on the enzymes that govern cholesteryl ester metabolism. Administration of a 20-mg bolus of lipoprotein cholesterol or a diet supplemented with 2% cholesterol resulted in an increase in microsomal cholesteryl ester content with little change in microsomal free cholesterol. This was accomplished by an increase in cholesteryl esterification as measured by ACAT but no change in CE hydrolase activity. An increased need for hepatic free cholesterol was experimentally induced by intravenous bile salt infusion or cholestyramine (3%) added to the diet. ACAT activity was decreased with both experimental manipulations compared to controls, while CE hydrolase activity did not change. Microsomal cholesteryl ester content decreased significantly with little change in microsomal free cholesterol content. Addition of exogenous liposomal cholesterol to liver microsomes from cholestyramine-fed and control rats resulted in a 784 +/- 38% increase in ACAT activity. Nevertheless, the decrease in ACAT activity with cholestyramine feeding was maintained. These studies allowed us to conclude that changes in hepatic free cholesterol needs are met in part by regulation of the rate of cholesterol esterification by ACAT without a change in the rate of cholesteryl ester hydrolysis by CE hydrolase.     

Studies on acyl-coenzyme A: cholesterol acyltransferase activity in human liver microsomes

The aim of the present study was to characterize the acyl-coenzyme A: cholesterol acyltransferase (ACAT) activity in human liver microsomes. Liver biopsies were obtained from patients undergoing elective cholecystectomy under highly standardized conditions. In 34 patients the enzyme activity of the microsomal fraction averaged 6.6 +/- 0.7 (mean +/- SEM) pmol.min-1.mg protein-1 in the absence of exogenous cholesterol. Freezing of the liver biopsy in liquid nitrogen increased the enzyme activity five- to sixfold. Similarly, freezing of the microsomal fraction prepared from unfrozen liver tissue increased the enzyme activity about twofold. These results may help to explain previous disparate results reported in the literature. The enhanced ACAT activity obtained by freezing was at least partly explained by a transfer of unesterified cholesterol to the microsomal fraction and possibly also by making the substrate(s) more available to the enzyme. Preincubation of the microsomal fraction, prepared from unfrozen liver tissue, with unlabeled cholesterol increased the enzyme activity about fivefold. This finding indicates that hepatic ACAT in humans can also utilize exogenous cholesterol as substrate. Addition of cholesterol to frozen microsomes prepared from unfrozen liver tissue increased the ACAT activity two- to threefold, whereas addition of cholesterol to microsomes prepared from frozen liver tissue did not further increase the enzyme activity. No evidence supporting the concept that ACAT is activated-inactivated by phosphorylation-dephosphorylation could be obtained by assaying the enzyme under conditions similar to those during which the human HMG-CoA reductase is inactivated-activated.     

Role of glycerol 3-phosphate and glycerophosphate acyltransferase in the nutritional control of hepatic triacylglycerol synthesis

1. Glycerol 3-phosphate content of isolated hepatocytes from starved rats and of glycogen-depleted hepatocytes from fed rats was low and severely limited triacylglycerol synthesis. 2. Raising the glycerol 3-phosphate content by addition of precursors to the cells resulted in a hyperbolic-like relationship between triacylglycerol synthesis and cellular glycerol 3-phosphate content. Statistical analysis of the curves showed no significant differences between the nutritional states either at saturating or at subsaturating glycerol 3-phosphate content. 3. V_max. of glycerophosphate acyltransferase measured in homogenized hepatocytes was decreased by 30–40% in starvation. There was no change in apparent K_m for glycerol 3-phosphate. Since at saturating glycerol 3-phosphate content esterification rates in hepatocytes of both nutritional states were identical, the enzyme is not limiting esterification under this condition. 4. At subsaturating glycerol 3-phosphate content the flux through glycerophosphate acyltransferase necessarily limits esterification. Therefore one would expect a decrease in esterification in starvation under this condition. This was the case when triacylglycerol synthesis was plotted against intracellular glycerol 3-phosphate concentration, calculated from the cellular glycerol 3-phosphate content and the intracellular water space, which was smaller in hepatocytes from starved rats. 5. The data obtained in hepatocytes were extrapolated to the intact liver by using the number of parenchymal cells per g of liver as determined from marker-enzyme analysis and the liver weight per 100g body weight. The extrapolation suggested that glycerol 3-phosphate is limiting esterification in vivo for contents below 0.3–0.4 and 0.5–0.65μmol/g for livers from fed and starved animals respectively. Also for a given fatty acid load and a glycerol 3-phosphate content below 0.3μmol/g the liver may esterify less in the starved state. However, at the glycerol 3-phosphate contents measured in freeze-clamped livers (0.30 and 0.44μmol/g for the fed and starved state respectively), livers in both nutritional states seemed capable of esterifying similar amounts of fatty acids.     

Perilipin A increases triacylglycerol storage by decreasing the rate of triacylglycerol hydrolysis

The perilipins are the most abundant proteins at the surfaces of lipid droplets in adipocytes and are also found in steroidogenic cells. To investigate perilipin function, perilipin A, the predominant isoform, was ectopically expressed in fibroblastic 3T3-L1 pre-adipocytes that normally lack the perilipins. In control cells, fluorescent staining of neutral lipids with Bodipy 493/503 showed a few minute and widely dispersed lipid droplets, while in cells stably expressing perilipin A, the lipid droplets were more numerous and tightly clustered in one or two regions of the cytoplasm. Immunofluorescence microscopy revealed that the ectopic perilipin A localized to the surfaces of the tiny clustered lipid droplets; subcellular fractionation of the cells using sucrose gradients confirmed that the perilipin A localized exclusively to lipid droplets. Cells expressing perilipin A stored 6-30-fold more triacylglycerol than control cells due to reduced lipolysis of triacylglycerol stores. The lipolysis of stored triacylglycerol was 5 times slower in lipid-loaded cells expressing perilipin A than in lipid-loaded control cells, when triacylglycerol synthesis was blocked with 6 microm triacsin C. This stabilization of triacylglycerol was not due to the suppression of triacylglycerol lipase activity by the expression of perilipin A. We conclude that perilipin A increases the triacylglycerol content of cells by forming a barrier that reduces the access of soluble lipases to stored lipids, thus inhibiting triacylglycerol hydrolysis. These studies suggest that perilipin A plays a major role in the regulation of triacylglycerol storage and lipolysis in adipocytes.     

Some properties and subcellular distribution of acyl-coenzyme A: retinol acyltransferase activity in rat testes

The present study was conducted to examine esterification of retinol by testicular microsomes. The microsomes were isolated from rat testes and were incubated under varying assay conditions with [3H]retinol. [3H]Retinylpalmitate was identified by reversed-phase high-performance liquid chromatography as an esterified product. The rate of esterification was increased by the addition of a fatty acyl-CoA. Coenzyme A esters of oleic, palmitic and stearic acids were equally effective substrates for retinol esterification. A 17-fold increase was observed in the presence of palmitoyl-CoA when microsomes had been pretreated with hydroxylamine, a reagent that reacts with coenzyme A thioesters to form hydroxamic acids. The esterifying activity was stimulated by the addition of dithiothreitol (4 mM) and fatty acid-free bovine serum albumin (1 mg/ml). The optimal concentrations for retinol and palmitoyl-CoA were 40 microM and 30-40 microM, respectively. The enzyme activity was inhibited by p-hydroxymercuribenzoate, sodium taurocholate and 5,5'-dithiobis-(2-nitrobenzoic acid), but not by EDTA. The enzyme activity was highest in microsomes (36%). However, some activity was present in mitochondria (29%). These results clearly show the presence of a fatty acyl-CoA: retinol acyltransferase that catalyzes the esterification of retinol in rat testes.     

Hepatic acyl-coenzyme A:cholesterol acyltransferase activity is decreased in patients with cholesterol gallstones

Altered hepatic cholesterol metabolism has been implicated in the etiology of cholesterol gallstones. This hypothesis has been examined by determining acyl-coenzyme A:cholesterol acyltransferase (ACAT) activity in liver biopsies from 31 cholesterol gallstone patients and 12 control subjects. Hepatic ACAT activity in gallstone patients was decreased to one-third that in controls (P less than 0.001). No differences in hepatic homogenate or microsomal free and total cholesterol concentrations were observed between the two groups. However, marked increases in free (107%) and total (98%) cholesterol concentrations were found in the cytosolic fraction of liver biopsies from gallstone patients. The total phospholipid concentration of the liver homogenate fraction was unchanged in both groups; however, the microsomal total phospholipid concentration was reduced by 17% (P less than 0.01) in gallstone samples compared with controls. This difference did not result in a significantly increased microsomal cholesterol/phospholipid ratio for the gallstone group (0.180 +/- 0.030) compared with the control group (0.169 +/- 0.042). These results show that hepatic ACAT activity is significantly decreased in cholesterol gallstone patients. These changes in ACAT activity in livers of patients with cholesterol gallstones are consistent with the known increase in the amount of free cholesterol secreted in the bile of these patients. Thus, the changes in ACAT activity may contribute to the pathogenesis of cholesterol gallstones.     

Serum activity and hepatic secretion of lecithin:cholesterol acyltransferase in experimental hypothyroidism and hypercholesterolemia

Lecithin:cholesterol acyltransferase (LCAT), the major cholesterol esterifying enzyme in plasma, plays an important role in the removal of cholesterol from peripheral tissues. This study in rat focuses upon the effects of hypothyroidism and cholesterol feeding on serum activity and hepatic LCAT secretion. To obviate the effect that inclusion of high concentrations of cholesterol in the rat serum may have on the proteoliposome used in the assay of LCAT, very low and low density lipoproteins (VLDL and LDL) were removed by ultracentrifugation at d 1.063 g/ml. The molar esterification rate in the euthyroid VLDL + LDL-free serum was found to be 0.94 +/- 0.06 compared to 0.67 +/- 0.05 in hypothyroid rats and 1.56 +/- 0.14 in hypercholesterolemic rats. LCAT secretion by suspension cultures of hepatocytes from hypercholesterolemic rats was found to be significantly depressed when compared to that for euthyroid and hypothyroid animals. Secretion by hepatocytes from hypothyroid rats was depressed for the first 0-4 hr, but rapidly recovered. The depressed secretion of LCAT by hepatocytes from hypercholesterolemic rats correlates with the appearance in the media of apoE-rich, discoidal HDL. Discoidal HDL was six times more effective as a substrate for purified human LCAT than HDL from hypercholesterolemic serum, and twice as effective as serum and nascent HDL from euthyroid animals. It is concluded that the depressed LCAT activity in serum from hypothyroid rats is due to a depressed hepatic secretion of the enzyme and that the elevated serum activity of hypercholesterolemic rats may be related to a defect in LCAT clearance. Finally, the appearance of discoidal HDL in the medium upon culture of hepatocytes from hypercholesterolemic rats appears to be due to an inhibition of LCAT secretion by these cells.     

Lipoic acid improves hypertriglyceridemia by stimulating triacylglycerol clearance and downregulating liver triacylglycerol secretion

Elevated blood triacylglycerol (TG) is a significant contributing factor to the current epidemic of obesity-related health disorders, including type-2 diabetes, nonalcoholic fatty liver disease, and cardiovascular disease. The observation that mice lacking the enzyme sn-glycerol-3-phosphate acyltransferase are protected from insulin resistance suggests the possibility that the regulation of TG synthesis be a target for therapy. Five-week-old Zucker Diabetic Fatty (ZDF) rats were fed a diet containing (R)-α-lipoic acid (LA, ∼200 mg/kg body weight per day) for 5 weeks. LA offset the rise in blood and liver TG by inhibiting liver lipogenic gene expression (e.g. sn-glycerol-3-phosphate acyltransferase-1 and diacylglycerol O-acyltransferase-2), lowering hepatic TG secretion, and stimulating clearance of TG-rich lipoproteins. LA-induced TG lowering was not due to the anorectic properties of LA, as pair-fed rats developed hypertriglyceridemia. Livers from LA-treated rats exhibited elevated glycogen content, suggesting dietary carbohydrates were stored as glycogen rather than becoming lipogenic substrate. Although AMP-activated protein kinase (AMPK) reportedly mediates the metabolic effects of LA in rodents, no change in AMPK activity was observed, suggesting LA acted independently of this kinase. The hepatic expression of peroxisome proliferator activated receptor α (PPARα) target genes involved in fatty acid β-oxidation was either unchanged or decreased with LA, indicating a different mode of action than for fibrate drugs. Given its strong safety record, LA may have potential clinical applications for the treatment or prevention of hypertriglyceridemia and diabetic dyslipidemia.     

Isolation and characterization of Chinese hamster ovary cell mutants deficient in acyl-coenzyme A:cholesterol acyltransferase activity

A protocol has been developed for isolating cholesterol ester-deficient cells from the Chinese hamster ovary cell clone 25-RA. This cell line previously was shown to be partially resistant to suppression of cholesterogenic enzyme activities by 25-hydroxycholesterol and to accumulate a large amount of intracellular cholesterol ester when grown in medium containing 10% fetal calf serum (Chang, T. Y., and Limanek, J. S. (1980) J. Biol. Chem. 255, 7787-7795). The higher cholesterol ester content of 25-RA is due to an increase in the rate of cholesterol biosynthesis and low density lipoprotein receptor activity compared to wild-type Chinese hamster ovary cells, and not due to an abnormal acyl-CoA:cholesterol acyltransferase enzyme. The procedure to isolate cholesterol ester-deficient mutants utilizes amphotericin B, a polyene antibiotic known to bind to cholesterol and to form pore complexes in membranes. After incubation in cholesterol-free medium plus an inhibitor of endogenous cholesterol biosynthesis, 25-RA cells were found to be 50-500 times more sensitive to amphotericin B killing than were mutant cells containing reduced amounts of cholesterol ester. Twelve amphotericin B-resistant mutants were isolated which retained the 25-hydroxycholesterol-resistant phenotype. These mutants did not exhibit the perinuclear lipid droplets characteristic of 25-RA cells, and lipid analysis revealed a large (up to 40-fold) reduction in cellular cholesterol ester. The acyl-CoA:cholesterol acyltransferase activities of these cholesterol ester-deficient mutants were markedly lower than 25-RA when assayed in intact cells or in an in vitro reconstitution assay. The tightest mutant characterized, AC29, was found to have less than 1% of the parental acyl-CoA:cholesterol acyltransferase activity. These mutants all have reduced rates of sterol synthesis and lower low density lipoprotein receptor activity compared to 25-RA, probably as a consequence of their reduced enzyme activities. Cell fusion experiments revealed that the phenotypes of all the mutants examined are not dominant and that the mutants all belong to the same complementation group. We conclude that these mutants contain a lesion in the gene encoding acyl-CoA:cholesterol acyltransferase or in a gene encoding a factor needed for enzyme production.     

Solubilization and purification of rat liver microsomal 1,2-diacylglycerol: CDP-choline cholinephosphotransferase and 1,2-diacylglycerol: CDP-ethanolamine ethanolaminephosphotransferase.

1. The procedure, which involved 2-step sonication of microsomes at pH 7.4 and then at pH 8.5 in the presence of sodium deoxycholate and subsequent dialysis, resulted in 4-5-fold purification of choline-phosphotransferase and ethanolaminephosphotransferase with the yield of 40-50%. 2. Ethanolaminephosphotransferase was further purified 8.5-fold over microsomes by sucrose density gradient centrifugation of the partially purified preparation, while cholinephosphotransferase activity was considerably lost during this procedure. No separation of the two transferases from each other was achieved at this step. 3. Cholinephosphotransferase required Mg2+ as cofactor, and microsomal phospholipids for its maximal activity. On the other hand, Mn2+ was more effective than Mg2+ as cofactor for ethanol aminephosphotransferase, and this enzyme was inhibited by microsomal phospholipids. 4. Both transferases were stimulated several-fold by sodium deoxycholate and also showed similar optimal pH ranging from pH 8.0 to 8.5. 5. Km values for 1,2-diacylglycerol emulsion were 81.0 muM for cholinephosphotransferase and 63.0 muM for ethanolaminephosphotransferase, respectively. CDP-choline and CDP-ethanolamine competitively inhibited, with the same Ki value (both 350 muM), ethanolaminephosphotransferase and cholinephosphotransferase, respectively. The Ki values obtained were much greater than the corresponding Km values for the cytidine substrates (36.4 muM for CDP-choline and 22.0 muM for CDP-ethanolamine). 6. The partially purified enzymes were further treated with Triton X-100. When enzyme activities were assayed with Mg2+, cholinephosphotransferase, although considerably inactivated, was partially separated from ethanolaminephosphotransferase by sucrose density gradient centrifugation of Triton-treated preparations. Furthermore, cholinephosphotransferase (but not ethanol-aminephosphotransferase) itself was partially separated into Mg2+ -requiring and Mn2+ -requiring components. In contrast, ethanolaminephosphotransferase assayed with either Mg2+ or Mn2+ formed a single peak together with Mn2+ -requiring cholinephosphotransferase.     

Recruiting a new substrate for triacylglycerol synthesis in plants: the monoacylglycerol acyltransferase pathway

Background

Monoacylglycerol acyltransferases (MGATs) are predominantly associated with lipid absorption and resynthesis in the animal intestine where they catalyse the first step in the monoacylglycerol (MAG) pathway by acylating MAG to form diacylglycerol (DAG). Typical plant triacylglycerol (TAG) biosynthesis routes such as the Kennedy pathway do not include an MGAT step. Rather, DAG and TAG are synthesised de novo from glycerol-3-phosphate (G-3-P) by a series of three subsequent acylation reactions although a complex interplay with membrane lipids exists.

Methodology/Principal Findings

We demonstrate that heterologous expression of a mouse MGAT acyltransferase in Nicotiana benthamiana significantly increases TAG accumulation in vegetative tissues despite the low levels of endogenous MAG substrate available. In addition, DAG produced by this acyltransferase can serve as a substrate for both native and coexpressed diacylglycerol acyltransferases (DGAT). Finally, we show that the Arabidopsis thaliana GPAT4 acyltransferase can produce MAG in Saccharomyces cerevisiae using oleoyl-CoA as the acyl-donor.

Conclusions/Significance

This study demonstrates the concept of a new method of increasing oil content in vegetative tissues by using MAG as a substrate for TAG biosynthesis. Based on in vitro yeast assays and expression results in N. benthamiana, we propose that co-expression of a MAG synthesising enzyme such as A. thaliana GPAT4 and a MGAT or bifunctional M/DGAT can result in DAG and TAG synthesis from G-3-P via a route that is independent and complementary to the endogenous Kennedy pathway and other TAG synthesis routes.     

Octanoate reduces very low-density lipoprotein secretion by decreasing the synthesis of apolipoprotein B in primary cultures of chicken hepatocytes

Fatty acids of varying lengths and saturation differentially affect plasma apolipoprotein B (apoB) levels. To identify the mechanisms underlying the effect of octanoate on very low-density lipoprotein (VLDL) secretion, chicken primary hepatocytes were incubated with either fatty acid-bovine serum albumin (BSA) complexes or BSA alone. Addition of octanoate to culture medium significantly reduced VLDL-triacylglycerol (TG), VLDL-cholesterol and apoB secretion from hepatocytes compared to both control cultures with BSA only and palmitate treatments, but did not modulate intracellular TG accumulation. However, no differences in cellular microsomal triglyceride transfer protein levels were observed in the cultures with saturated fatty acid. In pulse-chase studies, octanoate treatment resulted in reduced apoB-100 synthesis, in agreement with its promotion of secretion. This characteristic effect of octanoate was confirmed by addition of a protease inhibitor, N-acetyl-leucyl-leucyl-norleucinal (ALLN), to hepatocyte cultures. Analysis showed that the level of apoB mRNA was lower in cultures supplemented with octanoate than in the control cultures, but no significant changes were observed in the levels of apolipoprotein A-I, fatty acid synthase and 3-hydroxy-3-methylglutaryl-CoA reductase mRNA as a result of octanoate treatment. Time-course studies indicate that a 50% reduction in apoB mRNA levels requires 12 h of incubation with octanoate. We conclude that octanoate reduced VLDL secretion by the specific down-regulation of apoB gene expression and impairment of subsequent synthesis of apoB, not by the modulation of intracellular apoB degradation, which is known to be a major regulatory target of VLDL secretion of other fatty acids.     

Functionality of the seventh and eighth transmembrane domains of acyl-coenzyme A:cholesterol acyltransferase 1

Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1) is a resident enzyme in the endoplasmic reticulum. ACAT1 is a homotetrameric protein and contains nine transmembrane domains (TMDs). His460 is a key active residue and is located within TMD7. Human ACAT1 has seven free Cys, but the recombinant ACAT1 devoid of free Cys retains full enzyme activity. To further probe the functionality of TMD7 (amino acids 446-460) and TMD8 (amino acids 466-481), we used a parental ACAT1 devoid of free Cys as the template to perform Cys-scanning mutagenesis within these regions. Each of the single Cys mutants was expressed in Chinese hamster ovary (CHO) cell line AC29 lacking endogenous ACAT1. We measured the effect of single Cys substitution on enzyme activity and used the Cu(1,10-phenanthroline)2SO4-mediated disulfide cross-linking method to probe possible interactions of engineered Cys between the two identical subunits. The results show that several residues in one subunit closely interact with the same residues in the other subunit; mutating these residues to Cys does not lead to large loss in enzyme activity. Helical wheel analysis suggests that these residues are located at one side of the coil. In contrast, mutating residues F453, A457, or H460 to Cys causes large loss in enzyme activity; the latter residues are located at the opposite side of the coil. A similar arrangement is found for residues in TMD8. Thus, helical coils in TMD7 and TMD8 have two distinct functional sides: one side is involved in substrate-binding/catalysis, while the other side is involved in subunit interaction.     

Eicosapentaenoic acid reduces the invasive and metastatic activities of malignant tumor cells

Recent studies have shown that the cyclooxygenase and the 5-lipoxygenase pathways of arachidonic acid, are required for the invasive and metastatic activity of certain tumor cells. We show here that malignant murine melanoma and human fibrosarcoma cells cultured in media supplemented with eicosapentaenoic acid show a dose and time dependent decrease in invasiveness, in collagenase IV production and in the case of the murine cells, a reduced ability to metastasize to the lung after intravenous injection. It was also shown that a metabolite of eicosapentaenoic acid was less potent than the comparable arachidonic acid metabolite in restoring collagenase IV production and invasiveness after inhibition of the lipoxygenase pathway. These studies indicate that such supplements have the potential to reduce the metastasis of certain tumor cells, converting them to benign status.     

Chlamydia pneumoniae induces macrophage-derived foam cell formation by up-regulating acyl-coenzyme A: cholesterol acyltransferase 1

In macrophages, the accumulation of cholesteryl esters synthesized by acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) plays a crucial role in foam cell formation, a hallmark of early atherosclerotic lesions. It is suggested that Chlamydia pneumoniae (C. pneumoniae) induces foam cell formation. However, the mechanism of foam cell formation induced by C. pneumoniae has not been fully elucidated. In this study, we found that C. pneumoniae increased the expression of acyl-coenzyme A: cholesterol acyltransferase 1(ACAT1) mRNA and protein in a dose-dependent manner in THP-1-derived macrophages exposed to low density lipoprotein (LDL). In addition, C. pneumoniae dose-dependently suppressed the expression of peroxisome proliferator-activated receptor gamma (PPAR γ) mRNA and protein. Rosiglitazone, a specific PPAR γ agonist, not only dose-dependently alleviated the down-regulation of PPAR γ expression by C. pneumoniae infection, but also dose-dependently inhibited the C. pneumoniae-induced ACAT1 expression. Furthermore, higher doses of rosiglitazone (10 and 20 μM) suppressed the C. pneumoniae-induced foam cell formation from morphological (Oil red O staining) and biochemical (zymochemistry method) criteria. These results first demonstrate that C. pneumoniae induces macrophage-derived foam cell formation by up-regulating ACAT1 expression via PPAR γ-dependent pathway, which may contribute to its pro-atherogenic properties.     

Effect of gemfibrozil on triacylglycerol synthesis and secretion by liver and lipoprotein lipase activity in adipose tissue of rats

The role of gemfibrozil, a hypotriglyceridemic drug, in synthesis, secretion and catabolism of triacylglycerols (TG) in rats was assessed. Chow diet-fed Sprague-Dawley rats were given various doses of gemfibrozil (10, 30 and 100 mg/kg body weight) for 2 weeks. Rats receiving the drug at the lowest dose significantly lowered the concentration of serum TG and apolipoprotein (apo) B in comparison with control rats. Synthesis of fatty acids from [14C]acetate and esterification of [14C]oleate to TG by the liver were not suppressed by the drug. Secretion rates of TG and apo B, measured by the Triton method, were suppressed at the highest dose. Lipoprotein lipase activity of the acetone powder prepared from adipose tissue was not influenced by the drug. These results indicate that the primary cause of hypotriglyceridemic action of gemfibrozil is not due to suppressing synthesis and secretion of TG by the liver or enhancing lipoprotein lipase activity in adipose tissues.