Mechanism of activation of liver acetyl-coa carboxylase by cell swelling

Transcription

1 Eur. J. Biohem. 217, (1993) 0 FEBS 1993 Mehanism of ativation of liver aetyl-coa arboxylase by ell swelling Arnaud RAQUET', Viniane GAUSSIN', Mathieu BOLLEN2, Willy STALMANS' and Louis HUE' I Hormone and Metaboli Researh Unit, International Institute of Cellular and Moleular Pathology, and University of Louvain Medial Shool, Brussels, Belgium * Afdeling Biohemie, Faulteit Geneeskunde, Katholieke Universiteit Leuven, Belgium (Reeived July 26, 1993) - EJB /6 The ativation of hepati glyogen synthase by the amino-aid-indued ell swelling has been attributed to the stimulation of [glyogen-synthasel-phosphatase resulting from an inrease in the intraellular ontent in glutamate and aspartate, and a derease in intraellular C1-, whih is a ompensatory response to ell swelling [Meijer, A. J., Baquet, A., Gustafson, L., van Woerkom, G. M. & Hue, L. (1992) J. Bid. Chem. 267, Here we studied whether the ativation of aetyl-coa arboxylase by ell swelling ould be explained by the same mehanism. The ativation of endogenous or purified aetyl-coa arboxylase was measured in gel-filtered liver extrats or ytosols. No ativation ould be observed under basal onditions but a fivefold stimulation was obtained with onentrations of glutamate (20-25 mm) found in hepatoytes inubated with glutamine. A similar stimulation was also observed with other diarboxyli aids suh as malonate and suinate, or with metal ions like Mg2+, Ca2+ and Mn2+ (10 mm). The addition of mm C1- was found to inhibit the ativation of aetyl-coa arboxylase by some 20-30%. Mg2+ was also found to stimulate the ativation of the endogenous glyogen synthase. The glutamate-stimulated and Mg2+-stimulated ativation of glyogen synthase and aetyl-coa arboxylase was unaffeted by 10 pm inhibitor-2, a speifi inhibitory protein of protein phosphatase-1, but ould be nearly ompletely bloked by the phosphatase inhibitor miroystin-lr. Our data suggest that the amino-aid-indued ativation of aetyl-coa arboxylase and glyogen synthase in the liver ours by a ommon ioni mehanism. The stimulation of liver glyogen synthesis by amino aids, suh as glutamine, results from the ativation of glyogen synthase, the key enzyme in this pathway [l-21. This effet is mediated by an inrease in hepatoyte volume, whih results from the Na+-dependent entry of amino aids and the intraellular aumulation of atabolites like glutamate and aspartate [3, 41. A similar stimulation of glyogen synthesis and ativation of glyogen synthase is observed when hepatoytes are allowed to swell in a hypotoni medium, even in the absene of amino aids [3, 51. Glyogen synthase is not the only enzyme regulated by ell swelling and, among other effets, a stimulation of lipogenesis has also been reported [2, 6, 71. An ativation of aetyl-coa arboxylase (ACC), the key enzyme in fatty aid synthesis, is observed in swollen hepatoytes, whether swelling results from inubation in the presene of glutamine, or in a hypotoni medium [5]. The strong similarities between the ativation of both enzymes suggest that a ommon regulatory mehanism is involved [5]. Reently, we showed that the ativation of glyogen synthase by ell swelling results from the ellular response to Correspondene to L. Hue, Horn unit, ICP-UCL 7529, Avenue Hipporate 75, B-1200 Brussels, Belgium Abbreviations. ACC, aetyl-coa arboxylase ; PP, protein phosphatase. Enzymes. Aetyl-CoA arboxylase (EC ) ; [aetyl-coa arboxylase]-phosphatase (EC ) ; glyogen synthase (EC ); [glyogen-synthasel-phosphatase(ec ); protein phosphatase (EC ). the hange in volume [8]. To restore its initial volume, the swollen ell reats by extruding KC1 and, onsequently, the intraellular onentration of C1- dereases. Sine C1- inhibits synthase phosphatase [S], a derease in intraellular C1- onentration favours synthase ativation. Moreover, glutamate and aspartate, whih aumulate in hepatoytes inubated with glutamine [4, 61, were found to stimulate synthase phosphatase [S]. Therefore, the derease in C1-, together with the aumulation of glutamate and aspartate, stimulate synthase phosphatase and thus ativate glyogen synthase. The aim of this work was to study whether the mehanism by whih ell swelling stimulates synthase phosphatase ould also apply to the ativation of ACC. This would offer a ommon regulatory mehanism for the ativation of both synthase and ACC in hepatoytes inubated in presene of ertain amino aids. EXPERIMENTAL PROCEDURES Materials Gluagon (Novo Nordisk), miroystin-lr and okadai aid (Calbiohem), CNBr-ativated Sepharose 4B (Pharmaia), radiohemials (Amersham), and other biohemial reagents (Sigma or Boehringer Mannheim) were purhased as indiated. Purifiation of ACC, fatty aid synthase and inhibitor-2 ACC was purified from the livers of rats whih were starved for two days then refed with a low-fat, high-arbohy-

2 1084 drate diet for three days. They reeived 250 nmol/kg of gluagon intraperitoneally 15 min before sarifie to inativate their liver ACC. ACC was purified by frationation with poly(ethy1ene glyol) [9], followed by affinity hromatography on avidin sepharose [lo]. Two different preparations with speifi ativities (measured at 20 mm itrate) of about 30 mu/mg and 100 mu/mg protein, respetively, were used in this work. Fatty aid synthase was purified from the livers of refed rats (see above) aording to the method desribed in [ll]. The speifi ativity was 1.5 U/mg protein. Inhibitor- 2 was purified from rabbit skeletal musle [12]. Measurement of ACC Two methods were used to measure the ativity of ACC. The first one (method 1) measures the inorporation of 14Clabelled HC0,- into aid-soluble ompounds. This method is not speifi, sine it also measures the ativity of other arboxylases Therefore, it was only used with purified preparations of ACC. The assay was arried out as desribed [14], in 0.2 ml final volume and in the presene of 0.5 mm magnesium itrate. After 5 rnin inubation at 30 C, the reation was started by the addition of the enzyme (usually 20 pl) and proeeded linearly for up to 10 min. It was stopped after 10 min of inubation by the addition of 40 p1 6 M HCI. The inorporation of radioativity into aid-stable ompounds was measured as desribed [14]. The other method (method 2), whih is speifi, measures the inorporation of radioative aetyl units into lipids in the presene of an exess of purified fatty aid synthase. It was adapted from the method originally desribed by Bijleveld and Geelen for isolated hepatoytes [15] and was used in non-purified systems. The ativity was measured at 37 C in a final volume of 0.2 ml ontaining 60 mm Hepes, ph 7.5,2.5 mm EGTA, 0.25 mm dithioerythritol, 2 mm Mg- ATP, 10 mm KHCO?, 0.5 mm NADPH, 4.25 mg/ml fattyaid-free bovine serum albumin, 60 pm butyryl-coa, 60 pm [l-'4c]aetyl-coa (4Ci/mol), 3 mu of purified fatty aid synthase and, exept when otherwise stated, 0.5 mm magnesium itrate. The reation mixture was inubated for 5 rnin at 37"C, and the reation was started by the addition of 20 p1 of extrats and was linear for up to 6 min. The reation was stopped after 4 min by the addition of 0.1 ml 10 M NaOH. Saponifiation and extration of fatty aids were performed as desribed [ 151. The K, for itrate of ACC purified from the liver of gluagon-treated rats was measured by both methods and was 3 mm and 3.2 mm, whih is within the reported range of values (2-7 mm) [16, 171. Phosphorylation of ACC purified from rat mammary gland by CAMP-dependent protein kinase or by AMP-ativated protein kinase inativates ACC by dereasing the V, and inreasing the K, for itrate from about 2mM to 4mM or lomm, depending on the kinase [18, 191. Thus, 0.5 mm itrate, the onentration used in the assay, is below the reported K,. 1 U enzyme ativity orresponds to 1 pmol substrate transformedmin under the assay onditions. The proteins were measured [20] with bovine serum albumin as a standard. Measurement of the ativation of glyogen synthase and ACC The ativation of both glyogen synthase and ACC was measured in two different systems. In the first one, we used liver extrats as a soure of protein phosphatase. The substrates were the endogenous glyogen synthase, the endogenous ACC or 1 mu of added purified ACC. The liver extrats were prepared as follows. The livers from fed rats treated with gluagon (250 nmovkg intraperitoneally, 15 rnin before sarifie) were homogenized in a Potter-Elvehjem devie in 2.5 vol. of an ie-old homogenization buffer (0.1 M glyylglyine, ph 7.4, 0.25 M surose, 0.5 mm dithiothreitol). After entrifugation (8000 g, 10 min), 1 ml supernatant was filtered in the old through a olumn (9 ml) of Sephadex G-25 equilibrated in the homogenization buffer. The final onentration of liver proteins in the inubated extrats was about 15 mg proteidml, exept for the inubation with the protein phosphatase inhibitors, where the final onentration was mg protein/ml. The extrats were inubated immediately as indiated in the legends, and samples were withdrawn periodially for assays of glyogen synthase 121 and ACC (method 2) with 0.5 mm magnesium itrate. In the seond system, we used purified ACC as a substrate and a ytosoli fration as a soure of ACC phosphatase. The ytosoli fration was prepared by high-speed entrifugation ( g, 40 min) of a rat liver extrat (prepared as desribed above). This proedure indeed separates ACC phosphatase, whih has been reported to be present in the high-speed supernatant [21], from the bulk of [glyogen-synthasel-phosphatase, whih is known to be bound to, and reovered with, partiulate glyogen in the high-speed-entrifugation pellet [22]. We verified that more than 80% of the ACC phosphatase ativity was reovered in a high-speedentrifugation supematant (data not shown). The high-speedentrifugation supernatant was filtered through Sephadex G- 25 as desribed above. The inubation was performed in 50 mm glyylglyine, 125 mm surose, 0.5 mm dithiothreitol, with 1 mu/ml purified ACC, the indiated additions and 0.5 vol. of filtered high-speed-entrifugation supernatant (replaed by buffer when omitted). Samples were periodially withdrawn for the measurement of ACC (method 2). RESULTS Glutamate and Mg2+ stimulate ACC ativation in vitro The influene of C1-, glutamate and aspartate on glyogen-synthase ativation has been demonstrated in gel-filtered liver extrats in whih the ativation of the endogenous glyogen synthase was measured [81. The same system was used here as a first experimental approah to study the effets of these substanes on the ativation of the endogenous ACC. Rat liver extrats were filtered through Sephadex G-25 to remove the small-m, ligands. In suh a system, whih is devoid of Mg-ATP, protein kinases annot work and the timeourse of glyogen-synthase ativation an be readily measured 181. Fig. 1 shows that, in this system, the initial ativity of the endogenous ACC was very low as expeted, sine the rats were previously treated with gluagon to inativate ACC. When this extrat was inubated under basal onditions, there was very little, if any, ativation of ACC. This is at variane with glyogen synthase, whih is ativated under the same onditions [8]. By ontrast, the addition of 100 mm glutamate to the extrat indued a steady and linear ativation of ACC over a 1-h inubation period (Fig. 1A). The effet of glutamate on ACC ativation, measured over 45- rnin inubation periods, was dose dependent (Fig. 1 B). In the

3 B I Time of inubation (rnin) Conentration (mm) Fig. 1. Stimulation of ACC ativation by glutamate and Mg in liver extrats. Liver extrats were inubated at 25 C and samples were withdrawn at the indiated periods of time (A) or at 0 and 45 rnin (B) to measure the ativity of endogenous ACC by method 2. ACC ativation (8) represents the differene in ativity of ACC at 0 and 45 min. The values are the means 2SEM for three (A) or four (B) different preparations. Exept where indiated by (*), all the values are signifiantly different (P ) from those at time 0 (A) or at onentration 0 (B). (0), ontrol; (O), 100 mm (A) or the indiated onentration (B) of potassium glutamate; (A), 10 mm (A) or the indiated onentration (B) of magnesium aetate. presene of mm glutamate, onentrations found in hepatoytes inubated with 10 mm glutamine [4, 61, the ativity of ACC found after 45 rnin of inubation was about five-times that of the ontrol. The effet of Mg2+ was also tested in this system. Fig. 1A shows that the addition of 10 mm magnesium aetate ativated ACC after a lag period of about 10min; the same effet was obtained with MgCI, (data not shown). The effet was dose dependent, and half-maximal with 7-8 mm (Fig. 1B). C1- inhibits ACC ativation in vitro Sine there was no ACC ativation in gel-filtered liver extrats under basal onditions (Fig. 1 A), the possible inhibition of ACC ativation by C1- ould only be tested in the presene of a stimulator, e.g. 10 mm Mg. Under these onditions, the addition of CI- indued a dose-dependent inhibition of ACC ativation (Fig. 2). The inhibition was detetable for C1- onentrations greater than 30 mm, and, at 100 mm C1-, ACC ativation was inhibited by 32%. The effet of C1- was independent of the ounter-ation (K+ or Na ) used (data not shown). C1- was also able to inhibit the glutamatestimulated ativation of ACC. Indeed, in the presene of 25 mm glutamate, a 18?3% (n = 7) inhibition was observed with 100mM KCl (data not shown). It should be noted here that the inhibition of ACC ativation by C1- is muh smaller than that of glyogen-synthase ativation [8]. Indeed, in the same system, 50mM KC1 was able to ompletely inhibit the ativation of glyogen synthase [8], whereas a mere 20% inhibition of ACC ativation was observed (Fig. 2). Speifiity and nature of the effets of glutamate and Mgz+ The stimulation of ACC ativation by glutamate was also observed with other aids. The results, obtained with 50 mm salts, are shown in Fig. 3A. Aspartate, whih also aumulates in ells treated with glutamine [4, 61, stimulated ACC phosphatase. Both glutamate and aspartate are diarboxyli KCI onentration (rnm) Fig. 2. Inhibition of ACC ativation by C1- in liver extrats. Liver extrats were inubated at 25 C in the presene of 10 mm magnesium aetate and the indiated onentrations of KCI. Samples were withdrawn at 0 and 60 min to measure the ativity of endogenous ACC by method 2. ACC ativation represents the differene in ativity of ACC at 0 and 60 min. The values are the means +- SEM for nine different preparations. ACC ativation in the presene of Mg2+ alone (100%) was mu. h-l. g liver protein-. (*), signifiantly different (P ) from the mean value in absene of KC1. aids. Therefore, we tested whether other diarboxyli aids were able to mimi the stimulatory effet of glutamate. The most impressive stimulation of ACC phosphatase was obtained with suinate (60-fold stimulation) and malonate (100-fold stimulation), (Fig. 3 A). A dose-dependent stimulation of ACC ativation was also obtained with itrate, with a half-maximal effet (10-fold stimulation) at about 1 mm itrate (data not shown). Fig. 3A also shows that among the monoarboxyli aids tested, pyruvate was found to stimulate ACC phosphatase. A stimulation of ACC ativation ould also be observed with several divalent ations used at 10 mm (Fig. 3B). Ca and Mn2+ were better stimulators than Mg. In these experiments, a diret effet of the ligands on ACC ativity (rather than ativation) due to the transfer of ligands from the phosphatase inubation to the ACC assay

4 1086 Control K-oxalate K-propionate Na-suinate K-aspartate K-aetate Na-latate Control(]( B l a MgCI2 MnCI, COCI, ACC ativation (mu of ACC ativated. 60 min. g of liver protein- ) ACC ativation (mu of ACC ativated. 30 min-. g of liver protein- ) Fig. 3. Effet of monoarboxyli and diarboxyli aids (A) and divalent ations (B) on the ativation of ACC in liver extrats. Liver extrats were inubated at 25 C with or without the indiated additions. The onentrations of the salts were 50 (A) or 10 mm (B). Samples were withdrawn at 0 and 60 min (A) or 0 and 30 min (B) to measure the ativity of endogenous ACC by method 2. ACC ativation represents the differene in ativity of ACC at 0 and 60 (A) or 30 min (B). The values are the means 2 SEM for five (A) or three (B) different preparations. (*), Signifiantly different (P ) from ontrol values. 2oo Mg-ligand onentration (mm) Fig.4. Effet of itrate, malonate and glutamate on the ativity of purified ACC. The ativity of 1 mu purified ACC was measured by method 2 in the presene of the indiated onentrations of the Mgz+ salts of the indiated ligands. When malonate or glutamate were tested, no itrate was added in the assay. The values are the means 2SEM for three different experiments. The ativity is expressed as the perentage of the ativity measured in the presene of 20 mm magnesium itrate. (*), Signifiantly different (P 50.05) from values in the absene of salt. (O), Glutamate; (El), malonate; (m), itrate. an be eliminated beause of the 10-fold dilution of the extrats in the ACC assays, and mainly on the basis of the experiments desribed below (Fig. 4 and Table 1). However, the stimulation of ACC ativation by arboxyli aids ould result either from an enzyme-direted effet, i.e. a stimulatory effet on the phosphatase, or from a substrate-direted effet, rendering ACC a better substrate for the phosphatase. Ligands suh as itrate, malonate and C1- are indeed known to affet the ativity and/or the polymerization state of the enzyme [ To distinguish between these interpretations, we ompared the diret effet of glutamate and malonate on the ativity of purified ACC with that of itrate. Citrate was found to stimulate ACC ativity as expeted, whereas glutamate and malonate had little or no effet on this ativity (Fig. 4). Therefore, it is very unlikely that the ativation reported above for glutamate and malonate resulted from an effet on ACC itself. These results were further doumented by omparing the effets of glutamate and Mg on the ativity of purified ACC, whih had been inubated for 45 min with or without a filtered high-speed-entrifugation supernatant (Table 1). The 45-min inubation of ACC with glutamate or Mg2+, but without the high-speed-entrifugation supernatant, did not hange ACC ativity, thus ruling out an effet of these ions on the ativity of ACC itself. However, ACC ativation was well observed after the same period of inubation with the high-speed-entrifugation supernatant, as expeted. This indiates that proteins, probably protein phosphatase(s) were required to ativate ACC. Table 1 also shows that, in this partially purified system, C1- ould partially inhibit the glutamate-stimulated or Mg -stimulated ativation of ACC. The inhibition was obtained in the range mm KCI, onentrations found in and respetively 181. Effets of inhibitors of protein phosphatases The Ser/Thr-speifi protein phosphatases have been lassified in four major groups, aording to their substrate speifiity and the effets of inhibitors and ativators [28, 291. Type-1 protein phosphatases (PP-1) an be easily identified by their sensitivity to inhibition by two proteins, termed inhibitor-1 and inhibitor-2. The type-2 protein phosphatases (PP-2), whih are insensitive to these inhibitors, an be further differentiated into three groups aording to their requirement for ations. The type-2a phosphatases (PP-2A) do not require divalent ations, but their phosphorylase-phosphatase ativity is stimulated by polyations and by basi polypeptides. The type-2b phosphatases (PP-2B) or alineurins are Caz -dependent enzymes, while the type-2c phosphatases (PP-2C) represent Mg2+-dependent enzymes. In addition, PP-1 and PP-2A are inhibited by okadai aid or miroystin, whereas PP-2B is only weakly inhibited and PP-2C is not sensitive to these inhibitors [ Our previous investigations have shown that the glutamine-indued ativation of glyogen synthase and ACC in intat hepatoytes was inhibited by miroystin [5]. Miro-

5 Table 1. Effets of glutamate, MgZ+ and C1- on ACC ativity. Purified ACC (1 mu measured in the presene of 20 mm magnesium itrate) was inubated at 25 C in the absene or presene of a high-speed-entrifugation supematant for 0 or 45 rnin with the indiated additions before measuring its ativity by method 2. The values are the means tsem for (n) different preparations. (*), Signifiantly different (P 50.05) from the values in the presene of glutamate or Mg", but in the absene of KCl; (+), Signifiantly different (P 50.05) from the values with 15 mm KCl. n.d., not determined Additions ACC ativity without high-speed-entrifugation supernatant after inubation for with high-speed-entrifugation supernatant after inubation for 0 min (n = 4) 45 min (n = 4) 0 min (n = 4) 45 min (n = 4) % of added ativity Control mm glutamate 14f 4 13 t 4 +I5 mm KCl n.d mm KC1 n.d mm Mg mm KCl +40 mm KC t3 n.d. 323 n.d. 11 t f 9 11OOt 117 n.d. 1050? 115 n.d loo* n.d n.d f 230*.+ Gktamte # + A o-, * Control * _L1( Magnesium \ GUamte v Miroystin onentration (M) Fig. 5. Inhibition by miroystin of the glutamate-stimulated or Mgz+-stimulated ativation of glyogen synthase (A) and ACC (B) in liver extrats. Liver extrats were inubated at 25 C with the indiated onentration of miroystin, with no further addition (0), 100 mm potassium glutamate (0) or 10 mm magnesium aetate (A). Samples were withdrawn at 0 and 45 rnin to measure the ativities of endogenous glyogen synthase and ACC (method 2). Glyogen synthase and ACC ativations represent the differenes in ativity at 0 and 45 rnin. The values are the means?sem for three different preparations. (*), Signifiantly different (PSO.05) from values in the absene of miroystin; (Z), signifiantly different (P 50.05) from values in the absene of salt. ystin also bloked the glutamate-stimulated and Mg2+-stimulated ativation of glyogen synthase and ACC in diluted liver extrats (Fig. 5). In these diluted extrats, little ativation of the endogenous glyogen synthase was observed unless glutamate or Mg2+ was added (Fig. 5 and Table 2). The ativation of both substrates was half-maximally bloked by about 10 nm miroystin and no differene in sensitivity ould be deteted. We have also investigated whether PP- 1 ould be involved in the glutamate-stimulated and Mg2+stimulated ativation of glyogen synthase and ACC. The addition of high onentrations (10 pm) of inhibitor-2 did not affet the ativation of glyogen synthase or ACC in liver extrats (Table 2). We verified that a purified preparation of the atalyti subunit of PP-1 was indeed almost ompletely inhibited (90%) by only 10 nm inhibitor-2 (data not shown). DISCUSSION A ommon mehanism of ativation of ACC and glyogen synthase by ell swelling Hepatoyte swelling, indued by ertain amino aids or hypotoni media, ativates both glyogen synthase and ACC [5]. We have reently reported the mehanism of ativation of glyogen synthase by swelling [8]. In this report, we studied whether this mehanism ould apply for the ativation of ACC. In hepatoytes inubated with glutamine, the ativation of glyogen synthase an be explained by the fall in the intraellular onentration of C1-, whih inhibit synthase phosphatase, and the aumulation of glutamate and aspartate, whih stimulate synthase phosphatase [8]. We now find that the ativation of ACC in liver extrats filtered

6 1088 Table 2. Effet of inhibitor-2 on the glutamate-stimulated or Mg'+-stimulated ativation of glyogen synthase and ACC in liver extrats. Liver extrats were inubated for 20 rnin at 25 C with or without of 10 pm inhibitor-2 as indiated. Then, for synthase ativation, 100 mm potassium glutamate or 10 mh4 magnesium aetate were added and the extrats were further inubated for 8 min. Samples were withdrawn at 0 min and 8 min of inubation to measure the ativity of endogenous glyogen synthase a. Glyogen-synthase ativation represents the differene in ativity of the enzyme at 0 rnin and 8 min. For ACC ativation, 1 mu purified ACC was added after the initial inubation, together with 100 mm potassium glutamate or 10 mm magnesium aetate. The extrats were further inubated for 60 min at 30 C and samples were withdrawn at 0 rnin and 60 min of inubation to measure the ativity of ACC by method 1. ACC ativation represents the differene in ativity of ACC at 0 min and 60 min. The values are the means? SEM for three (glyogen synthase) or five (ACC) different experiments. (*), Signifiantly different (P ) from the values without salt. Conditions Synthase ativation with ACC ativation with ontrol inhibitor-2 ontrol inhibitor-2 U synthase a formed. 8 min-'. g liver protein-' U ACC ativated. h-'. g liver protein-.' ~. No salt 0.2? ? ? 0.3 Potassium glutamate 1.6*? * * I+_ *? 0.9 Magnesium aetate 3.8*? "? * *? 0.4 through Sephadex is greatly stimulated by glutamate and other diarboxyli aids as well as by Mg". The ACC ativation is also found to be slightly inhibited by C1-. Moreover, the 4-5-fold stimulation of the in vitro ativation of ACC by mm glutamate, together with the 20-30% stimulation resulting from a derease in the C1- onentration from 50 mm to 15 mm, are suffiient to explain the in vivo ativation obtained in hepatoytes inubated with 10 mm glutamine. Indeed, mu ACC/g protein are found in extrats inubated for 45 min, whih is quite similar to the mu ACC/g wet ells (i.e mu ACC/g protein) found in hepatoytes treated with 10 mm glutamine for min [5]. These effets are very reminisent of those involved in the mehanism of ativation of glyogen synthase by swelling [S]. Therefore, we propose that the fall in intraellular C1- onentration as well as the inrease in intraellular glutamate onentration, that are observed in swollen hepatoytes in the presene of glutamine [4, 6, 81, stimulate both [glyogen-synthasel-phosphatase and ACC phosphatase. A differene between these effets an however be observed, sine the effet of C1- is greater than that of glutamate on glyogen-synthase ativation, whereas the opposite is true for the ativation of ACC. In hepatoytes that are allowed to swell under hypotoni onditions and in the absene of amino aids, the ativation of ACC did not differ from that found in hepatoytes inubated with glutamine [5]. Glutamate, whih does not aumulate in ells inubated in hypotoni media, annot be involved in the stimulation of the phosphatase. Moreover, the fall of the intraellular onentration of C1-, whih was the same as in hepatoytes inubated with glutamine [S], is probably insuffiient to explain the in vivo ativation. Indeed, the stimulation of ACC phosphatase resulting from the derease in C1- is relatively small (20-25%). Therefore, other mehanism(s) should be involved for the ativation of ACC in hepatoytes inubated in hypotoni media. The finding that ACC ativation is stimulated by Mgz' in vim offers another regulatory mehanism, whih is urrently being tested in our laboratory. Nature of the protein-phosphatase(s) involved in the ativation of glyogen synthase and ACC Previous studies have shown that PP-1, PP-2A and PP- 2C aount for nearly all the synthase phosphatase and ACC- phosphatase ativities in subellular liver frations [ The analysis and the omparison of the properties of the glutamate-stimulated and Mg2+-stimulated phosphatase reported in this work with those of known protein phosphatases should allow us to identify the phosphatase involved. The glutamate-stimulated and Mg2+-stimulated ativation of glyogen synthase and ACC was not affeted by inhibitor-2, a speifi inhibitor of PP-1. The adopted onentration of inhibitor-2 (10 pm) was suffiiently high to inhibit all known type-1 protein phosphatases, inluding the glyogen-assoiated phosphatase that shows very poor sensitivity to inhibitor-2 [22]. The inhibition by miroystin exludes a role for PP-2C, whih is insensitive to this inhibitor. Similarly, the lak of dependene on Ca", eliminates PP-2B. Therefore, at this stage, the only argument to attribute the glutamatestimulated and Mg2'-stimulated phosphatase to PP-2A is by exlusion of the other known protein phosphatases. The formal identifiation of the protein phosphatase involved awaits further purifiation and haraterization. Surprisingly, the stimulatory effet of glutamate and Mg2' on the ativation of glyogen synthase was only obtained with the endogenous substrate (data not shown), while the ativation of ACC ould be easily demonstrated with both the endogenous and a purified substrate. This explains why the stimulatory effet of Mgz+ was not previously noted when purified glyogen synthase was used as a substrate [33]. It should also be pointed out here that the ontribution of PP-1, PP-2A and PP-2C to the ativation of glyogen synthase in subellular liver frations is variable and depends for example on the nature of the substrate and the assay onditions [29, 331. In onlusion, our data suggest that the amino-aid-indued ativation of ACC and glyogen synthase ours by a ommon ioni mehanism. This mehanism is mediated by protein phosphatase(s) with properties that seem to be different fom those of PP-1, PP-2B and PP-2C. We speulate that the protein phosphatase(s) involved in the ativation of both ACC and glyogen synthase belong(s) either to PP-2A or to the rapidly growing list of novel Ser/Thr-speifi protein phosphatases [34]. We thank M. H. Rider for ritial reading of the manusript. M. B. is Onderzoeksleider of the Nationaal Fonds voor Wetenshappelijk Onderzoek (Belgium). V. G. is a fellow of the Institutpour 1'Enouragement de la Reherhe Sientifque dans 1 'Industrie et l'agri-

7 1089 ulture (Belgium). This work was supported by the Belgian State- Prime Minister s Offie-Siene Poliy Inentive Program in Life Sienes 99/93-122, grant 20, and by grants from the Belgian Fund for Medial Sientifi Researh. REFERENCES 1. Katz, J., Golden, S. & Wals, P. A. (1979) Glyogen synthesis by rat hepatoytes, Biohem. J. 180, Lavoinne, A., Baquet, A. & Hue, L. (1987) Stimulation of glyogen synthesis and lipogenesis by glutamine in isolated rat hepatoytes, Biohem. J. 248, Baquet, A., Hue, L., Meijer, A. J., van Woerkom, G. M. & Plomp, P. 3. A. M. (1990) Swelling of rat hepatoytes stimulates glyogen synthesis, J. Biol. Chem. 265, Plomp, P. J. A. M., Boon, L., Caro, L. H. P., van Woerkom, G. M. & Meijer, A. J. (1990) Stimulation of glyogen synthesis in hepatoytes by added amino aids is related to the total intraellular ontent of amino aids, Eul: J. Biohem. 191, Baquet, A,, Maisin, L. & Hue, L. (1991) Swelling of rat hepatoytes ativates aetyl-coa arboxylase in parallel to glyogen synthase, Biohem. J. 278, Baquet, A., Lavoinne, A. & Hue, L. (1991) Comparison of the effets of various amino aids on glyogen synthesis, lipogenesis and ketogenesis in isolated rat hepatoytes, Biohem. J. 273, Haussinger, D. & Lang, F. (1991) Cell volume in the regulation of hepati funtion: a mehanism for metaboli ontrol, Biohinz. Biophys. Ata 1071, Meijer, A. J., Baquet, A., Gustafson, L., van Woerkom, G. M. & Hue, L. (1992) Mehanism of ativation of liver glyogen synthase by swelling, J. Biol. Chem. 267, Moore, F., Weekes, J. & Hardie, D. G. (1991) Evidene that AMP triggers phosphorylation as well as diret allosteri ativation of rat liver AMP-ativated protein kinase. A sensitive mehanism to protet the ell against ATP depletion, EUK J. Biohem. 199, Tipper, J. P. & Witters, L. A. (1982) In vitro phosphorylation and inativation of rat liver aetyl-coa arboxylase purified by avidin affinity hromatography, Biohim. Biophis. Ata 715, Linn, T. C. (1981) Purifiation and rystallization of rat liver fatty aid synthetase, Arh. Biohem. Biophys. 209, Yang, S.-D., Vandenheede, J. R. & Merlevede, W. R. (1981) A simplified proedure for the purifiation of the protein phosphatase modulator (inhibitor-2) from rabbit skeletal musle, FEBS Lett. 132, Davies, D. R., Van Shaftingen, E. & Hers, H.-G. (1982) Interferene by pyruvate arboxylase in the measurement of aetyl-coa arboxylase in rude liver preparations, Biohem. J. 202, Holland, R., Witters, L. A. & Hardie, D. G. (1984) Gluagon inhibits fatty aid synthesis in isolated hepatoytes via phosphorylation of aetyl-coa arboxylase by yli-amp-dependent protein kinase, Eul: J. Biohem. 140, Bijleveld, C. & Geelen, M. J. H. (1987) Measurement of aetyl- CoA arboxylase ativity in isolated hepatoytes, Biohim. Biophys. Atu 918, Numa, S. & Yamashita, S. (1974) Regulation of lipogenesis in animal tissues, Cum Top. Cell. Regul. 8, Kim, K. H. (1979) Control of aetyl-coa arboxylase by ovalent modifiation, Mol. Cell. Biohem. 28, Hardie, D. G. & Guy, P. S. (1980) Reversible phosphorylation and inativation of aetyl-coa arboxylase from latating rat mammary gland by yli AMP-dependent protein kinase, Eul: J. Biohem. 110, Davies, S. P., Sim, A. T. R. & Hardie, D. G. (1990) Loation and funtion of three sites phosphorylated on rat aetyl-coa arboxylase by the AMP-ativated protein kinase, Eul: J. Biohern. 187, Bradford, M. M. (1976) A rapid and sensitive method for the quantitation of mirogram quantities of protein utilizing the priniple of protein-dye binding, Anal. Biohem. 72, Witters, L. A. & Baon, G. W. (1985) Protein phosphatases ative on aetyl-coa arboxylase phosphorylated by asein kinase 1, asein kinase I1 and the CAMP-dependent protein kinase, Biohem. Biophys. Res. Commun. 130, Bollen, M., Vandenheede, J. R., Goris, J. & Stalmans, W. (1988) Charaterization of glyogen-synthase-phosphatase and phosphorylase phosphatase in subellular liver frations, Biohim. Biophys. Atu 969, Gregolin, C., Ryder, E. & Lane, M. D. (1968) Liver aetyl oenzyme A arboxylase. I. Isolation and atalyti properties, J. Biol. Chem. 243, Gregolin, C., Ryder, E., Warner, R. C., Kleinshmidt, A. K., Chang, H. C. & Lane, M. D. (1968) Liver aetyl oenzyme A arboxylase. 11. Further moleular haraterization, J. Bid. Chem. 243, Lane, M. D., Moss, J., Ryder, E. & Stoll, E. (1971) The ativation of aetyl CoA arboxylase by triarboxyli aids, Adv. Enzyme Regul. 9, Allred, J. B. & Roehrig, K. L. (1980) Inhibition of rat liver aetyl CoA arhoxylase by hloride, J. Lipid Res. 21, Kim, K. H., Lopez-Casillas, F., Bai, D. H., Luo, X. & Pape, M. E. (1989) Role of reversible phosphorylation of aetyl-coa arboxylase in long-hain fatty aid synthesis, FASEB J. 3, Cohen, P. (1989) The struture and regulation of protein phosphatases, Annu. Rev. Biohem. 58, Bollen, M. & Stalmans, W. (1992) The struture, role, and regulation of Type 1 protein phosphatases, Crit. Rev. Biohem. Mol. Biol. 27, MaKintosh, C., Beattie, K. A., Klumpp, S., Cohen, P. & Codd, G. A. (1990) Cyanobaterial miroystin-lr is a potent and speifi inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants, FEBS Lett. 264, Honkanen, R. E., Zwiller, J., Moore, R. E., Daily, S. L., Khatra, B. S., Dukelow, M. & Boynton, A. L. (1990) Charaterization of miroystin-lr, a potent inhibitor of type 1 and type 2A protein phosphatases, J. Biol. Chem. 265, Cohen, P., Holmes, C. F. B. & Tsukitani, Y. (1990) Okadai aid: a new probe for the study of ellular regulation, Trends Biohem. Si. IS, Stalmans, W. & Bollen, M. (1987) The determination of glyogen-synthase-phosphatase ativity : importane of the substrate and the type of assay, Adv. Protein Phosphutuses 4, Cohen, P. T. W., Brewis, N. D., Hughes, V. & Mann, D. J. (1990) Protein serinekhreonine phosphatases ; an expanding family, FEBS Lett. 268,