Relationship between the inhibition of phosphatidic acid phosphohydrolase-1 by oleate and oleoyl-CoA ester and its apparent translocation

Relationship between the inhibition of phosphatidic acid phosphohydrolase-1 by oleate and oleoyl-CoA ester and its apparent translocation

Biochimie 87 (2005) 437–443 www.elsevier.com/locate/biochi Relationship between the inhibition of phosphatidic acid phosphohydrolase-1 by oleate and ...

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Biochimie 87 (2005) 437–443 www.elsevier.com/locate/biochi

Relationship between the inhibition of phosphatidic acid phosphohydrolase-1 by oleate and oleoyl-CoA ester and its apparent translocation N. Elabbadi a,b,*, C.P. Day b, A. Gamouh a, A. Zyad a, S.J. Yeaman b a

Laboratoire d’Immunologie, Biochimie et Biologie Moléculaire, Faculté des Sciences et Techniques, Université Cadi Ayyad, B.P. 523 Beni-Mellal, Morocco b School of Biochemistry and Genetics and Center for Liver Research, Medical School, University of Newcastle upon Tyne, Newcastle upon Tyne NE2 4HH United Kingdom Received 8 April 2004; accepted 4 January 2005 Available online 02 February 2005

Abstract Phosphatidic acid phosphohydrolase-1 (PAP-1) activity is reversibly inhibited by fatty acids and their acyl-CoA esters and it appears paradoxical that these effectors have been reported to increase the liver’s esterification capacity by translocating the rate-limiting enzyme PAP-1 from cytosol to the endoplasmic reticulum. Therefore, we have examined the effect of oleate, oleoyl-CoA, and spermine on the activation and translocation of PAP-1 of rat liver. PAP-1 activity is directly inhibited by oleic acid and oleoyl-CoA ester in an allosteric manner, resulting in the formation of inactive PAP-1-fatty acid (or -acyl-CoA) complex, even in the absence of any subcellular structures. Such association/aggregation of PAP-1 can be easily collected by centrifugation and may explain the apparent translocation phenomenon of this enzyme to a particular structure in the presence of fatty acids or acyl-CoA esters as reported in many works. Indeed, incubation of cytosol fraction alone with oleate or oleoyl-CoA at 37 °C, followed by centrifugation, induces a significant increase (sevenfold) in PAP-1 activity in the pellet fraction. This displacement is accompanied by an increase in the specific activity of PAP-1 in the pellet fraction. Spermine is less effective than oleate in inducing the displacement of PAP-1 activity from cytosol to the pellet fraction in the absence of any membrane structures. This apparent translocation of PAP-1 is also promoted when homogenate fraction was incubated with oleate prior to the preparation of cytosol and microsomal fraction. Thus, many of the announced factors, including fatty acids, would promote the in vitro association/aggregation of PAP-1 enzyme rather than its translocation, and therefore, re-evaluation of the reported effects on PAP1 translocation phenomenon is required. It is proposed that fatty acids and their esters would favour b-oxidation over esterification by promoting the forming of inactive associated PAP-1 in situations such as starvation and metabolic stress in which there is an increased supply of fatty acids to the liver. © 2005 Elsevier SAS. All rights reserved. Keywords: Phosphatidic acid phosphohydrolase; Regulation; Fatty acids; Rat liver

1. Introduction In mammalian cells, two forms of phosphatidic acid phosphohydrolase (PAP) activity have been identified [1,2]. PAP-1, stimulated by Mg2+ and inhibited by N-ethylmaleimide

Abbreviations: BSA, bovine serum albumin; DAG, sn-1,2-diacylglycerol; ER, endoplasmic reticulum; NEM, N-ethylmaleimide; PA, phosphatidic acid; PAP, phosphatidic acid phosphohydrolase; TAG, triacylglycerol. * Corresponding author. Tel.: +212 23 48 51 12; fax: +212 23 48 52 01. E-mail address: [email protected] (N. Elabbadi). 0300-9084/$ - see front matter © 2005 Elsevier SAS. All rights reserved. doi:10.1016/j.biochi.2005.01.003

(NEM), is distributed between the cytosol and endoplasmic reticulum (ER) and is regarded as the metabolic form, involved in the regulation of glycerolipid synthesis. In contrast, PAP-2 is Mg2+-independent and NEM-insensitive and is found mainly in the plasma membrane and in view of its location, is thought to be involved in cell-signalling [3,4]. The metabolic expression of PAP-1 activity is thought to be regulated by the ability of the cytosolic form to translocate to the ER, where its substrate, phosphatidic acid (PA), is generated mainly by the acylation of glycerol-3-phosphate [5,6]. Largely indirect evidence suggests that this translocation is

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induced by the accumulation of free fatty acids, acyl-CoA esters, and PA on the ER membranes, resulting in the enhanced synthesis of glycerolipids [6–10]. Polyamines such as spermine and spermidine have been used to promote the in vitro translocation of PAP-1 to the microsomal membranes, and their effect being synergistic with that of fatty acids [11,12]. On the other hand, amphipatic cationic compounds, such as chlorpromazine, decrease the rate of sn-1,2diacylglycerol (DAG) formation in a cell free system of rat liver by displacing PAP-1 from microsomal membranes and by preventing its interaction with these membranes [6]. Such interpretation has come essentially from experiments that have been carried out by incubating cells or cell-free tissue homogenates with lipids or other compounds prior to the preparation of microsomal and cytosol fractions by centrifugation and/or filtration methods [6,12,13]. Recently, we showed that PAP-1 activity is directly inhibited by fatty acids and their acyl-CoA esters [14] and as such they cannot promote directly neither the activation, nor the translocation of this enzyme to the ER as reported in many studies [5,6,9,10,13]. Here, we show that fatty acids and their acyl-CoA esters induce the formation of inactive PAP-1-fatty acids (or acyl-CoA) complex, even in the absence of any subcellular structures. Such association/aggregation of PAP1 enzyme can be easily collected by centrifugation and may explain the apparent translocation process in the presence of fatty acids, acyl-CoA, or spermine. This study supplies new information on the possible physiological interaction of fatty acids and their esters with PAP-1 enzyme. 2. Materials and Methods Male Wistar rats (150–200 g) were obtained from Newcastle University Comparative Biology Centre (United Kingdom). They were allowed free access to food and water prior to sacrifice by cervical dislocation, after which livers were immediately removed, and stored at –20 °C until use. PA, sn-1,2-dioleoylglycerol, oleic acid, oleoyl-CoA, bovine albumin serum free fatty acids, Triton X-100, chlorpromazine, spermine, and polyethylene glycol (MW 8000) were from Sigma Chemical (Poole, Dorset, United Kingdom). 2.1. Preparation of subcellular fractions from rat liver Livers from rats were suspended at 4 °C in 4–5 volumes of 50 mM Tris–HCl buffer (pH 7.4) containing 0.25 M sucrose and 0.2 mM dithiothreitol (buffer A) and minced with scissors before homogenisation with a Teflon-glass homogeniser. The crude homogenate was then centrifuged at 4 °C for 20 min at 10,000 × g and the supernatant (homogenate) collected. The homogenate was then centrifuged at 150,000 × g for 90 min at 4 °C, the resulting supernatant being recentrifuged for a further 60 min at 150,000 × g to produce a cytosolic fraction. For partial purification of PAP-1, cytosol fraction was diluted in buffer A to give a final protein concentration of

6–7 mg/ml, and then solid polyethylene glycol (MW 8000) was added (6 g per 100 ml of cytosol). The mixture was stirred for 1 h at 4 °C and then centrifuged at 5000 × g for 15 min. The precipitate (in which PAP-1 is enriched approximately five- to 10-fold) was resuspended in buffer A. Contamination of this fraction with PAP-2 was negligible, as judged by the complete sensitivity of the activity to NEM. 2.2. Determination of PAP activity This was measured by the production of water soluble [32P]inorganic phosphate from the substrate [32P]PA, which was synthesized as reported previously [15]. PA was dispersed by sonication (bath water sonicator) in buffer A until the solution becomes clear. Unless otherwise specified, the incubation mixture (0.1 ml) contained 50 mM Tris–HCl, pH 7.5, 1.5 mM MgCl2, 0.1 mg/ml fatty acid-free BSA, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM dithiothreitol, 125 mM sucrose (basic assay system), and 0.5 mM [32P]PA 10,000 cpm/nmol. After 10 min incubation at 37 °C, the reaction was terminated by the sequential addition of 0.25 ml of 1 M HCL in methanol, 0.25 ml chloroform, and 0.25 ml of 1 M MgCl2. After vortexing, the phases were separated by centrifugation (10,000 × g, 30 s) and a 0.3 ml aliquot of aqueous layer was transferred to a scintillation vial and radioactivity was counted in 4 ml of liquid scintillation. The time of the reaction and amount of enzyme protein added to the reaction mixture were chosen to ensure that no more than 10% of PA was hydrolysed to DAG. One unit of PAP activity was defined as the release of 1 nmol of [32P]inorganic phosphate per minute. 2.3. Effects of oleate, oleoyl-CoA, spermine, chlorpromazine, and DAG on PAP activity Aliquots of partially purified PAP was assayed as described above except that oleate, oleoyl-CoA, spermine, chlorpromazine, DAG or mixed oleate, plus Triton X-100 (0.3%) were present in the basic assay system. In some experiments, after 10 min preincubation with oleate, various concentration of BSA, PA, or Triton X-100 were added to the incubations 5 min prior to the initiation of PAP assays. Oleate, oleoyl-CoA, and PA were dispersed by sonication (bath water sonicator) in buffer A until the solution becomes clear. Suitable concentrations were then prepared by dilution. Displacement of PAP-1 activity from cytosol to the pellet fraction. Samples of homogenate or cytosol fraction were first incubated for 10 min at 37 °C in the presence of oleate, oleoylCoA, spermine or mixed spermine, plus oleate in a final volume of 0.35 ml. The mixtures were then cooled in ice before centrifugation for 1 h at 100,000 × g at 4 °C. The resulting pellets and aliquots (10 µl) from each resulting supernatant were then mixed with 0.2 ml of 50 mM Tris–HCl buffer (pH 7.5) containing 4 mg/ml fatty acid-free BSA, 2.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA and 1 mM dithiothreitol (buffer B) for 5 min at 37 °C. PAP activity was then measured by

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adding 0.05 ml of 2 mM [32P]PA to each mixture for a further 10 min incubation at 37 °C.

3. Results A number of studies have been shown that the in vitro translocation of PAP-1 from cytosol to membrane fraction is dependent to the presence of fatty acids, acyl-CoA esters, and polyamines such as spermine [7,8,11,12]. To determine whether or not the presence of oleate, oleoyl-CoA, and spermine have effects on the physical properties of PAP-1, we first examined the direct effect of these compounds on partially purified PAP-1. Fig. 1 shows that oleate and oleoylCoA progressively inhibited PAP-1 activity as we have reported previously [14]. In contrast, the product of PAPmediated hydrolysis, DAG, which has been shown to inhibit rat liver PAP-2 activity [16] was found to cause a slight activation of PAP-1 activity. This slight activation by DAG is more likely to be due to the detergent effect of DAG rather than its specific effect on cytosolic PAP form. Spermine at low concentrations (0.1–1 mM) was found to activate PAP1 activity with a 2.2-fold increase in this activity at 0.3 mM. Chlorpromazine was also found to activate PAP-1 with an effective concentration at 2 mM. These findings are in agreement with previous works [17,18]. The reversibility of oleate-induced inhibition of PAP1 was investigated by addition of PA (from 0.5 to 1.5 mM) or BSA (from 0.1 to 15 mg/ml), following preincubation of partially purified PAP-1 with 1 mM oleate but prior to assaying PAP activity. The presence of 1 mM oleate in the preincubation period reduced PAP-1 activity by more than 80%. When PA was added to reverse the inhibition effect of oleate, the

Fig. 1. Effect of oleate, oleoyl-CoA, DAG, spermine and chlorpromazine on phosphatidic acid phosphohydrolase-1 activity. Activity of partially-purified PAP from cytosol (specific activity 20 U/mg) was measured in the presence of various concentrations of oleate (·), oleoyl-CoA (_), DAG (L), spermine (*) and chlorpromazine (♦) as indicated. Each value is the mean ± S.E.M. for triplicate determinations. Results are expressed as % of control (without compounds).

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PAP-1 activity was restored progressively until complete reversion, which is obtained at about 0.7 mM PA (not shown). BSA also progressively reversed the inhibitory effect of oleate on this activity and complete reversion was reached at about 4–5 mg/ml of BSA (not shown). The addition of Triton X-100 to the mixture which had been preincubated with 0.6 mM oleate did not reverse the inhibitory effect of this fatty acid on PAP-1 activity, even at high concentrations of Triton X-100 (0.1–1%) (not shown). In contrast, if Triton X-100 is added simultaneously with oleate to the reaction mixture, this inhibition was completely cancelled. This is shown in Fig. 2 in which the initial velocity of PAP-1 activity vs. PA was measured in the presence of either oleate or oleate together with Triton X-100. In the absence of inhibitor, the activity was found to exhibit a typical hyperbolic binding curve, whereas in the presence of oleate (negative effector) the enzyme displayed a sigmoidal curve as reported in our previous work [14]. On the other hand, if 0.3% Triton X-100 is present in the reaction mixture, the effect of 0.6 mM oleate was completely abolished, even at very low concentrations of PA. These results indicate that PAP1 activity is inhibited by fatty acids and activated by PA and rule out the possible detergent effects of fatty acids on this activity. 3.1. Subcellular distribution of PAP-1 activity in the presence of oleate, oleoyl-CoA, spermine, and/or PA To further elucidate the possible mechanism of fatty acidand their acyl-CoA-induced PAP-1 inhibition, aliquots of cytosolic fraction were centrifuged, following preincubation with various compounds as indicated (Table 1). To reverse the inhibitory effect of oleate and oleoyl-CoA, pellets as well as the resulting supernatant fractions were mixed with buffer B containing 4 mg/ml of fatty acid-free BSA before PAP activ-

Fig. 2. Effect of PA on the initial velocity of phosphatidic acid phosphohydrolase-1 in the presence of oleate and Triton X-100. Activity of partiallypurified PAP from cytosol (specific activity 20 U/mg) was measured as a function of PA concentrations in the absence (·) or the presence of 0.6 mM oleate (*) or 0.6 mM oleate plus 0.3% Triton X-100 (_) in the reaction mixture. Each value is the mean ± S.E.M. for triplicate determinations.

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Table 1 Displacement of phosphatidic acid phosphohydrolase-1 activity from cytosol to the pellet fraction by oleate, oleoyl-CoA and spermine. Cytosol fraction obtained after centrifuging homogenate fraction from rat liver for 90 minutes at 150,000 × g (see Methods) was re-centrifuged once more for 1 hour at 150,000 × g at 4°C. Samples of the resulting supernatant (0.3 ml, 25 mg protein/ml) were pre-incubated for 10 min at 37°C in the presence of oleate, oleoyl-CoA, spermine or mixed oleate and spermine as indicated in a final volume of 0.35 ml. The mixtures were cooled in ice and centrifuged for 1 hour at 100,000 × g at 4°C. Separately, pellets and 10 µl of the resulting cytosol were mixed with 0.2 ml of buffer B containing 4 mg/ml BSA free fatty acids and incubated for 5 min at 37°C, then 0.05 ml of 2 mM [32P]PA was added to each mixture for a further ten minutes incubation at 37°C. Each value is the mean ± S.E.M. for triplicate determinations Additions None Oleate 0.25 mM Oleate 0.5 mM Oleate 1 mM Oleoyl-CoA 0.25 mM Oleoyl-CoA 0.5 mM Oleoyl-CoA 1 mM Spermine 1 mM Spermine 1 + oleate 0.5

Distribution of PAP activity (units per fraction) Pellet Cytosol 0.22 ± 0.04 13.6 ± 1 1.34 ± 0.09 11.6 ± 0.75 1.6 ± 0.08 11.4 ± 0.6 1.1 ± 0.1 12.5 ± 0.85 0.2 ± 0.05 13.3 ± 0.75 0.25 ± 0.04 13.3 ± 1.1 0.52 ± 0.04 13 ± 0.8 0.7 ± 0.1 11.88 ± 1.4 1.43 ± 0.12 11.07 ± 1

ity was assayed. The addition of 0.25, 0.5, or 1 mM oleate to the incubation induced a significant displacement of PAP1 activity from the cytosol to insoluble fraction (pellet) when compared with a control to which no fatty acid had been added. At the effective oleate concentration of 0.5 mM, the PAP activity in the pellet fraction was increased sevenfold. Oleoyl-CoA was less effective than oleate in inducing the displacement of PAP activity to the pellet fraction. Addition of 1 mM spermine also increased the PAP activity in the pellet fraction threefold, while the addition of 1 mM spermine together with 0.5 mM oleate further increased the activity by about 6.5-fold associated with the pellet fraction. There was no synergistic effect between spermine and oleate on displacement of PAP-1 activity from the cytosol to the pellet fraction. Similar results were found when the experiment was carried out with partially purified PAP-1 from the cytosol (not shown). In all cases, the displacement of PAP-1 activity from the cytosol fraction to the pellet fraction was accompanied by an increase in the specific activity of PAP-1 (not shown), demonstrating this is a specific effect on PAP-1. This indicates strongly that the effect of fatty acids and their acyl-CoA esters may act by a mechanism involving negative allosteric binding, resulting in an aggregation of PAP-1 enzyme-fatty acid complex. Also, the addition of oleate (0.5 mM) to homogenate fraction followed by incubation at 37 °C induced a significant displacement of PAP activity from cytosol to microsomal frac-

Distribution (%) Pellet/Cytosol 1.6/98.4 10.4/89.6 12.3/87.7 8/92 1.4/98.6 1.8/98.2 3.8/96.2 5.5/94.5 10.5/89.5

Total PAP activity (units per fractions) cytosol + pellet 13.82 ± 1.04 12.94 ± 0.76 13 ± 0.68 13.6 ± 0.95 13.5 ± 0.8 13.55 ± 1.14 13.52 ± 0.84 12.58 ± 1.3 12.37 ± 1.12

tion when compared with a control to which no fatty acid had been added (Table 2). As expected the addition of 0.5 mM of PA did not affect strongly the effect of oleate (0.5 mM), since PAP-1 was inhibited by oleate with a Ki value of 50 ± 10 µM, which is three times lower to the value of Km (130 ± 20 µM) [14].

4. Discussion One of the major levels of acute regulation of phosphatidic acid phosphohydrolase-1 (PAP-1) activity has been reported to be its translocation to the membranes and this intracellular movement is thought to be controlled by fatty acids [7,8]. Polyamines, like spermine or spermidine, have also been shown to promote this translocation phenomenon and to be synergistic with fatty acids [11,12]. Here, we demonstrate clearly that oleate induces the formation of inactive PAP-fatty acids complex at concentrations well below the critical micelle concentration. As such it cannot promote directly neither the activation, nor the translocation of this key enzyme. Thus, all the experiments carried out previously in this field showed that incubations of cells (hepatocytes, adipocytes) or cell-free tissue homogenates with fatty acids or their acyl-CoA esters induce the displacement of PAP1 to the pellet fraction after centrifugation or filtration. Only these techniques do not allow distinguishing between a true

Table 2 Distribution of phosphatidic acid phosphohydrolase activity between the cytosol and the microsomal fraction in the presence of oleate. Homogenate fractions (42 mg protein/ml) were preincubated for ten minutes at 37°C in the presence of oleate or mixed oleate and PA as indicated in a final volume of 0.35 ml. The mixtures were cooled in ice/water, centrifuged at 100,000 × g for 1h at 4°C and the resulting pellets and cytosol fractions (10 µl) were mixed with 0.2 ml of buffer B containing 4 mg/ml BSA free fatty acids for five minutes at 37°C. PAP activity was then measured by adding 0.05 ml of 2 mM [32P]PA to each mixture for a further ten minutes incubation at 37°C. Each value is the mean ± S.E.M. of triplicate determinations. Additions None at 37°C 0.5 mM oleate 0.5 mM PA 0.5 mM PA + 0.5 mM oleate

Distribution of PAP activity (units per fraction) Cytosol Microsomal 13 ± 1 9 ± 0.4 6.4 ± 0.5 16.1 ± 1 12.7 ± 0.75 9.5 ± 0.4 7 ± 0.3 15.7 ± 0.7

Distribution (%) Cytosol/Microsomal 59/41 28.5/71.5 57/43 31/69

Total activity (units per fractions) Cytosol + Microsomal 22 ± 1.4 22.5 ± 1.5 22.2 ± 1.15 22.7 ± 1

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specific translocation and sedimentation of inactive PAP-1fatty acids complex. Indeed, incubation of cytosol or partially purified PAP-1 with oleate, oleoyl-CoA, or spermine followed by centrifugation induced the displacement of PAP1 activity to the pellet fraction in the absence of any membrane structures (Table 1). This displacement was accompanied by an increase in the specific activity of PAP-1 in the pellet fraction. Such association/aggregation of PAP-1 enzyme can be easily collected by centrifugation and may explain the observed translocation process, since the effects of fatty acids and their acyl-CoA esters on subcellular distribution of PAP1 between a soluble (cytosolic) and a membrane fraction (microsomal or mitochondrial) have been measured mainly by using centrifugation or filtration methods [2,8,9,11,13, 19,20]. Therefore, caution must be used when interpreting these data, since the regulation of PAP-1 by fatty acids and their acyl-CoA esters involves a negative allosteric interaction, leading to the formation of inactive PAP-1-fatty acids (or -acyl-CoA esters) complex rather than its translocation. On the other hand, fatty acid- or acyl-CoA-induced PAP1 translocation in intact cells [7] could be an indirect effect resulting from their incorporation into newly synthesized PA, which has been shown to promote the binding of PAP-1 to microsomal and mitochondrial membranes in vitro [21,22]. The levels of added fatty acids and acyl-CoA esters used in our studies are consistent with those used previously in similar work [7,9,17] but are some what higher than values that are normally considered to be physiological. Fatty acids and their acyl-CoA esters would have detergent effects at high concentrations and such effects can lead to the enzyme inhibition. However, the most experimental concentrations of oleate (0–0.6 mM) used in the present study are much lower than the critical micelle concentration of oleate (from 0.72– 3.5 mM [23]). This suggests that the oleate is able to inhibit PAP-1 when it is in the monomeric form. It is also important to point out, that the inhibitory effects of fatty acids are greater at lower levels of PA (Fig. 2) and the concentrations of fatty acids actually present in the incubations will be lower than the added amounts owing to the presence of low levels of fatty acid-free BSA. Addition of detergent Triton X-100 up to 1% to the mixture which had been preincubated with oleate (0.6 mM) did not reverse the inhibitory effect of this fatty acid on PAP1 activity (not shown), suggesting highly that Triton X-100 alone is unable to dissociate PAP-fatty acids complex. On the other hand, additions of Triton X-100 (0.3%) simultaneously with oleate (0.6 mM) to the reaction mixture overcome any inhibition of PAP-1 activity, even at very low concentrations of PA (Fig. 2). Since the critical micelle concentration of this detergent is low (0.015%), the majority of oleate would be associated with Triton X-100 mixed micelles [24]. The formation of Triton X-100/oleate mixed micelles may cause surface dilution of monomeric oleate, thus preventing oleate from inhibiting PAP-1. Taking all of the above considerations into account, we conclude that the inhibitory effect of oleate on PAP-1 activity can only be

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explained by a direct effect on the enzyme, leading to the formation of inactive PAP-1-fatty acids complex, and rule out the possible detergent effects of fatty acids on this activity, at least by relatively low (0.1–0.6 mM) concentrations of fatty acids. Polyamines (polycationic substances) are present in almost all living cells and their concentrations in the cells are subjected to rapid change under various physiological and pathological conditions [25]. In our study, we confirm that spermine activates PAP-1 (Fig. 1), suggesting the possibility that polyamines may be involved in the regulation of this key enzyme. But our results argue against their involvement in a translocation process of PAP-1, since spermine can cause a non-specific sedimentation of the soluble PAP activity (Table 1) as observed previously [26]. The free intracellular concentration of fatty acids and their acyl-CoA esters are strictly modulated by specific binding protein (FABP, ACBP) and the free concentration of liver cytosolic long-chain acyl-CoA will not exceed 0.2 µM under normal physiological conditions [27]. Nevertheless, it cannot be excluded, that large local changes in the free concentration of long-chain fatty acids and their acyl-CoA esters may occur, for example in the mitochondria, where the total level of acylCoA can increase to extremely high levels (1 mM) [28]. Similarly, when hepatocyte cells are exposed to the high concentration of fatty acids, the free cytosolic concentration of fatty acids is probably higher than that of acyl-CoA, reaching the mM range [29,30]. During starvation and metabolic stress, the liver well receive high levels of fatty acids from the portal vein, where they will be mainly directed to the supply of energy (b-oxidation) rather than their incorporation into glycerolipids. In such conditions, PAP-1 activity (Ki for oleate ≈ 50 µM) should be considerably slowed down and points out the possibility that fatty acids and their acyl-CoA esters could act as regulatory molecules of PAP-1 activity in vivo. Although it is premature to extrapolate in vivo the aggregation phenomenon obtained in vitro, the proposed model for PAP-1 regulation involving association/dissociation is interesting, since the active form of PAP-1 (dissociated form) is induced by PA while the inactive form (associated PAP form) is induced by fatty acids and their acyl-CoA esters, and thereby, PAP-1 activity could be regulated by the intracellular content of membrane-bind PA or free PA and the availability of fatty acids (esterified or not) as we have suggested previously [14]. Furthermore, this inhibitory effect of oleate and oleoyl-CoA on PAP-1 activity seems to be in full agreement with the function of the glucose/fatty acid cycle. Indeed, in fed state, the high ratio of insulin to glucagon enhances glucose uptake and oxidation in the liver but does not inhibit hepatic fatty acid uptake. Increased hepatic glucose oxidation in turn inhibits fatty acids oxidation by blocking their entry into mitochondria via elevation of malonyl-CoA levels, and diverts fatty acids toward esterification. The subsequent increase in PA on the ER will then activate PAP-1 and overcome any fatty acid- and/or acyl-CoA-induced inhibition of PAP-1 activity, ending in an accelerated synthesis of glycero-

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lipids. On the other hand, the increased ratio of glucagon to insulin (e.g., starved state and during metabolic stress) directs incoming fatty acids (derived from adipose tissue lipolysis) toward b-oxidation by inhibiting acetyl-CoA carboxylase activity, leading to a reduction in malonyl-CoA levels and stimulation of carnitine palmitoyl transferase, thus promoting the entry of fatty acids into mitochondria. According to our model, the concomitant inhibition of PAP-1 activity by fatty acids and their acyl-CoA esters should favour the supply of energy. A number of studies have shown that the availability of fatty acids to the liver is an important regulatory of triacylglycerol (TAG) synthesis [31–35], while the accumulation of TAG in the liver is a process common in many disease states, such as thermal injury [36,37], obesity [38], diabetes [39], hepatitis C infection [40], and critical illness [36,37,41], and in pregnancy [42]. PAP-1 is known to primarily involved in controlling cellular DAG that acts as the precursor for TAG pool, as well as a substrate for phosphatidylcholine and phosphatidylethanolamine biosynthesis in the Kennedy pathway [43,44]. Therefore, regulation of this key enzyme plays a crucial role in determining the rate and direction of glycerolipid synthesis. This is supported by the finding that elevated levels of PAP activity being associated with alcoholic fatty liver [45]. In rat adipocytes, the inhibitory effect of catecholamines (noradrenaline) on PAP-1 activity was concomitant with increased levels of fatty acids. This effect is antagonised by insulin and b-adrenergic antagonist [46,47]. In the current study, the observed correlation between bromoethanol lactone-induced apoptosis and the strong inhibition of PAP1-regulated events, stress the key role of this enzyme in the integrity and the survival of the cell [48]. However, to what extent mammalian PAP-1 could be involved in the pathophysiology of hepatic steatosis mentioned above has yet to be elucidated.

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This work was supported by a project grant from the Biotechnology and Biological Sciences Research Council, Swindon, United Kingdom.

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