The EMBO Joumal Vol.1 No.6 pp.687-692, 1982

Differentiation of Ob17 preadipocytes to adipocytes: requirement of

adipose conversion factor(s) for fat cell cluster formation

Paul Grimaldi, Philippe Djian, Raymond Negrel, andGerard Ailhaud*Centre de Biochimie (LP 73-00), Universite de Nice, Parc Valrose,06034 Nice, France

Communicated by R. Levi-MontalciniReceived on 17 May 1982

An active fraction present in fetal calf serum (adipose conver-sion factor(s) or ACF) controls the formation of fat cellclusters observed during the adipose conversion of Ob17 cells.This conclusion is based upon microscopic examinations,autoradiographic experiments, and activity levels of charac-teristic enzyme maikers. ACF has no effect on the doublingtime of exponentially-growing cells but is required in the firstfew days of the resting phase. ACF has been partiallypurified; it is a low mol. wt. component ( < 6000-8000), pro-tease-insensitive, thermostable at neutral pH but not atstrongly acidic pH. An active fraction recovered from bovinepituitary extract shows properties similar to those of ACF.Therefore, ACF plays the role of a mitogenic factor specificfor cells susceptible to conversion to adipose cells.Key words: preadipocytes/adipose conversion/triiodo-thyronine/adipogenic factors

IntroductionThe formation of fat cell clusters is ubiquitous during the

differentiation of established preadipocyte cell lines. Thiscluster formation is due to the existence of a limited numberof post-confluent mitoses which affect cells susceptible to adi-pose conversion (Pairault and Green, 1979; Hiragun et al.,1980; Djian et al., in preparation). The Ob17 cell line used inthis study has been established from the epididymal fat pad ofthe C57 BL/6J ob/ob mouse (Negrel etal., 1978). Differentia-ted Ob17 cells present both morphological and biochemicalproperties characteristic of mature adipocytes. Prolonged ex-

posure to insulin at supraphysiological concentrations exerts a

mitogenic effect on Ob17 cells but when present at physio-logical concentrations exerts only potent lipogenic effects (P.Grimaldi et al., unpublished data). We reported recently thatadipose conversion of Ob17 cells is accelerated by physiologi-cal concentrations of triiodothyronine (71) (Gharbi-Chihi etal., 1981). The role of T3 is to amplify the phenotypic expres-sion of the differentiation program at the cellular level. Dur-ing the course of this investigation indirect evidence suggestedthat fetal calf serum (FCS), treated in order to remove thy-roid hormones, was lacking some other component(s) re-

quired for the formation of fat cell clusters. The characteriza-tion and the biological effects of this active fraction ("adiposeconversion factor(s)" or ACF), as well as its partialpurification, are described here. ACF, which is also present in

bovine pituitary extract, is shown to control post-confluentmitoses of susceptible cells and thus the formation of fat cell

clusters.

*To whom reprint requests should be sent.

( IRL Press Limited, Oxford, England. 0261-4189/82/0106-0687$2.00/0.

ResultsAdipose conversion in the presence of resin-treated serum

Confluent cells were maintained in the presence of treatedor untreated FCS, with or without T3. As Table I clearly in-dicates, chronic exposure to T3 causes a significant increase inthe specific activities of the enzyme markers of adipose con-version, as already described (Gharbi-Chihi et al., 1981).However, cells exposed to resin-treated serum in the presenceor, even more so, in the absence of T3, attain levels of activitysignificantly lower than those of control experiments (untrea-ted serum + Ta), either for different enzymes at a given time(day 9 post-confluence) or for a single enzyme (acid:CoAligase) as a function of time in culture. Lactate dehydrogenase(LDH) activity, not directly related to lipogenesis and to tri-glyceride synthesis, remains at similar levels under all condi-tions. Oil Red 0 staining of 18-day post-confluent cells(Figure 1) is in agreement with the data of Table I. Fat ac-cumulates in cells maintained in the presence of untreatedserum but does not accumulate significantly in cells exposedto resin-treated serum. In both cases a positive effect of T3 isobserved. Altogether these results suggested that resin-treatedserum is lacking component(s) other than T3 which appears tobe essential for the optimal expression of the different enzymephenotypes and for fat accumulation. These components ap-pear to be non-essential for exponentially-growing cells sincean identical doubling time is obtained with untreated andresin-treated serum (Figure 2). Moreover, resin-treated serumis able to support cell growth with the same doubling timeduring at least 13 passages at a 1:10 dilution.Requirement of a serum fraction for adipose conversionThe requirement for a factor (or factors) absent in the

resin-treated serum was determined using two different ap-proaches (Figure 3). First, a fraction - referred to as ACF -

was recovered (see Materials and methods) and added to con-fluent cells maintained in the presence of resin-treated serum(Figure 3A). Second, different proportions of resin-treatedand untreated sera were used (Figure 3B). The results indicatethat increasing the proportion of untreated serum increases,in a parallel fashion, the specific activities of two marker en-zymes, acid:CoA ligase and glycerol-3-phosphate dehydro-genase (G.3.PDH). In both experiments no saturation can bereached, suggesting that the concentration of this factor inserum is limiting for adipose conversion. The action of thisfactor appears to be general on adipose conversion, since thespecific activities of the two key enzymes, acid:CoA ligaseand G.3.PDH, involved respectively in triglyceride synthesisand in glucose utilization are increased in parallel. For themost active preparations ofACF, at any given time after con-fluence, 0.4 jig of active fraction per ml of culture mediumcontaining resin-treated serum, leads to enzyme activitiesequivalent to those obtained in 10% untreated FCS. Thisvalue corresponds on a protein basis to a 1200-fold purifica-tion.As expected, and in agreement with results of Figure 3, in-

clusion of the active fraction in resin-treated serum and T3restores formation of fat cell clusters to the extent observed in

687

P. Gimiddi et d.

Table I. Activity levels of differentiation markers of Obl7 cells exposed to resin-treated serum

Acid:CoA ligase G.3.PDH DGATa FAS ATP- LDHcitratelyase

Days after confluence 3 6 9 15 9 9 9 9 9

Untreated FCS 0.7 3.17 6.33 13.54 365 2.53 1.92 11.42 3220Untreated FCS + T3 0.7 - 9.7 19.2 383 3.72 2.6 14 3120Resin-treated FCS 0.69 1.8 1.5 3.47 69 0.44 0.5 3.31 2870Resin-treated FCS + T3 0.64 - 4.4 9.8 148 1.73 1.17 6.58 2800

Obl7 cells were grown in the presence of 1007o untreated FCS and shifted at confluence to four sets of conditions in the presence of 170 nM insulin and,where indicated, of 1.5 nM T3. Assays were performed in triplicate from two pooled culture dishes. All activities are expressed in mU/mg of protein (seeMaterials and methods).aDGAT, diglyceride:acylCoA acyltransferase.

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Fig. 1. Oil Red 0 staining of late confluent cells maintained in resin-treatedor untreated serum. 4 x 104 Obl7 cells were seeded in 60 mm diameterculture dishes and grown in 10%7o resin-treated FCS. They were shifted atconfluence to six sets of conditions as indicated, in the presence of 170 nMinsulin. When added, T3 and ACF were present at 1.5 nM and 0.4 itg/mlof culture medium, respectively. Cells were stained for lipids with Oil RedO 18 days after confluence.

cells exposed to untreated serum supplemented with T3(Figure 1). It is worth noting that these experiments show thatno lipid accumulation is visible in cells in the presence ofACFbut in the absence of T3. This result demonstrates that T3 isessential for the phenotypic expression of the differentiationprogram of 0b17 cells.Specific effect ofACF on post-confluent mitoses

Recent experiments on 0b17 cells have shown that, asalready described for 3T3/F442A cells in suspension (Pairaultand Green, 1979), the early period (3-10 days) of the restingphase is characterized by a wave of mitoses which involvesspecifically susceptible cells (P. Djian et al., in preparation).[3H]Thymidine incorporation and autoradiographic experi-ments demonstrate that the developing labeled cell clustersare those which are found later as fat cell clusters, and thatpost-confluent labeling outside clusters remains low.

Data reported in Table II indicate that, in contrast to con-trol cells maintained in untreated serum (experiment 1), DNAsynthesis is absent in cells maintained in resin-treated serum688

Fig. 2. Growth curves of Obl7 cells in media supplemented with 10%resin-treated (O 0) or 1007o untreated serum (0 0). Eachreported value is the mean of triplicate dishes. Variations between assaysdid not exceed 6%.

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Fig. 3. Effects of ACF on the levels of acid:CoA ligase and G.3.PDHactivities. A: Cells were grown (35 mm diameter dishes; 2 ml of culturemedium) in 10%o untreated FCS and shifted at confluence to 10% wesin-treated FCS containing 170 nM insulin, 1.5 nM T3, and increasing concen-trations of ACF. The active fraction was obtained as described in Materialsand methods and contained 0.085 mg/ml of protein. B: Confluent cellswere exposed to variable proportions of resin-treated or untreated FCS inthe presence of 170 nM insulin and 1.5 nM T3. In both cases assays wereperfonned 12 days after confluence and enzyme activities expressed as inTable I. Treat., resin-treated serum; untr., untreated serum.

(experiment 2). Inclusion of T3 has no effect on DNA synthe-sis (experiment 3), while chronic exposure to T3 and ACF in

12r 11

Adipose conversion factorf(s)

Table II. Specific effects of ACF on post-confluent mitosis

Experiment Culture Number of labeled Number of labeled Labeling indexnumber conditions dusters/dish nuclei/cluster in clusters outside cluster

I Untreated 40 20-60 50-60%0 12%oserum + T3

2 Resin-treated 0 - - 7%oserum

3 Resin-treated 0 - - 8%7oserum + T3

4 Resin-treated 31 20-40 50-70%o 8%oserum + ACF + T3

In experiment 1, Obl7 cells were grown and maintained in the presence of untreated serum. At confluence, 17 nM insulin and 1.5 nM T3 were added. In ex-periments 2-4, cells were grown and maintained in resin-treated serum and supplemented at confluence with 17 nM insulin as indicated. [3H]Thymidine in-corporation was performed on 6-day post-confluent cells and autoradiographs analyzed as described in Materials and methods.

A

D

a

E

c

F

H

Fig. 4. Micrographs of late confluent cells treated with ACF and/or T3.4 x 10' cells (first series) in 60 mm diameter culture dishes (A- F) or I0Ocells (second series) in 35 mm diameter culture dishes (G-1) were seededand grown in 1007o resin-treated FCS. They were shifted at confluence tonine different conditions in the presence of 170 nM insulin. Microscopicexaminations were performed 18 days (A- F) or 13 days (G -1) after con-fluence under direct light transmission. Micrographs were chosen asrepresentative areas for each series of cels. Adipose cells appear as whitespots on a dark field. The micrograph in (B) was intentionally overexposedin order to reveal dusters of cells which do not contain lipid droplets andwhici are not stainable with Oil Red 0 (see Figure 1). (A) Confluent cellsmaintained in resin-treated FCS; (B) cells as in (A) plus ACF (0.4 itg/mlculture medium); (C) cells as in (A) plus 1.5 nM T3; (D) cells as in (A) plusACF (0.4 4g/ml) and 1.5 nM T3; (E) cells in untreated FCS plus 1.5 nMT3; (F) cells as in (D) but ACF (0.4 ug/ml) was treated at strongly acidicpH (see Results); (G) cells as in (C) but using another batch of resin-treatedserum; (H) cells as in (G) plus pituitary extract (I ytg/ml culture medium);(1) cells as in (G) plus a fraction obtained from pituitary extract as describ-ed in Materials and methods. The protein concentration of this fractionwas too low to be determined accurately. Magnification x 16.

resin-treated serum restores the formation of labeled cell clus-ters to the level determined for control cells (experiment 4).Thus, the effect ofACF on DNA synthesis seems specific forsusceptible cells since no labeled cell cluster could be detectedin its absence, while labeling indices outside labeled clustersremain similarly low under all conditions. When confluentObl7 cells are maintained in resin-treated serum supplement-ed with 17 nM insulin and 1.5 nM T3, bovine pituitary extractor the active fraction purified from the pituitary extract, in-

duce, similarly to ACF, the formation of labeled cell clusters(not shown).ACF and theformation offat cell clusters

Micrographs presented in Figure 4 are in agreement withthe data of Table II. Confluent Ob17 cells maintained inresin-treated serum do not convert significandly to adiposecells (Figure 4A). Inclusion ofACF in the absence of T3 hasno visible effect on adipose conversion but causes the forma-tion of "undifferentiated" clusters, in which the cells do notcontain lipid droplets (Figure 4B) and at high magnificationthey appear less attached to the substratum. When chronic-ally exposed to T3 in resin-treated serum, in the absence ofACF, Ob17 cells do convert to adipose cells but differentiatedcells are present either as individual cells or in clusters con-taining a few cells (Figure 4C). Chronic exposure to T3andheat-treated ACF of cells maintained in resin-treated serumleads to a restoration of adipose conversion in fat cell clusters(Figure 4D) similar to that observed for control cells (Figure4E, untreated serum and T). It is of interest that ACF,before trypsin treatment or before heat treatment, presents alower activity, suggesting that it could bind to some otherserum component(s) or that either proteolytic or heat treat-ment causes an inactivation of some inhibitor(s) of ACF ac-tion. ACF remains active over a large range of pH (2.5 -8.0)for 2 h at 20°C as well as at pH 7.0 for 10 min at 90°C. Inac-tivation does occur at more acidic pH and a high temperature(0.5 N HCI for 15 min at 90°C), resulting in the loss of its ac-tivity in the formation of large fat cell clusters. However, fatclusters of small size or single adipose cells remain present(Figure 4F). Substitution of ACF by bovine pituitary extractshows that the extract also contains a factor(s) which plays arole in the formation of fat cell clusters (Figure 4H comparedto Figure 4G). A fraction can be obtained from bovine pitui-tary extract according to the procedure used for the purifica-tion of ACF from FCS (see Materials and methods). Thisfraction is also found to be active in the formation of fat cellclusters (Figure 4I).

Therefore, the role of ACF, which can be replaced by apituitary extract, is to increase the proportion of adipose cellsrelative to non-adipose cells while T3 acts at the level of eachindividual cell. This two-stage amplification phenomenon issupported by the data of Figure 5. ACF added to resin-treatedserum is not able to increase significantly the specific activitylevels of enzyme markers. T3 alone causes a more potent ef-fect. A combination of ACF and T3 is required to restore en-

689

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P. Gnmidi etal.

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Fig. 5. Dependence of adipose conversion upon T3 and ACF or pituitaryextract. Two different series of cells (A and B) were grown in lO7o untrea-ted FCS and shifted at confluence to resin-treated FCS supplemented as in-dicated in the presence of 170 nM insulin. The enzyme activities weredetermined 16 days after confluence and the results are expressed as inTable I. When present, the concentrations of ACF and T3 were 0.4 yg/mlculture medium and 1.5 nM respectively. The pituitary extract was obtain-ed as described in Materials and methods. l0(07o values correspond tothose obtained in the presence of untreated FCS plus 1.5 nM T3 and areequal to 830 and 790 mU/mg for G.3.PDH in A and B, respectively, to12.2 mU/mg for acid:CoA ligase and to 1.6 mU/mg for DGAT.

zyme activity levels similar to those obtained in control cells(untreated serum plus T3). As expected, acid-treated ACF isfound to be inactive and enzyme activity levels decrease tothose determined in resin-treated serum plus T3. Replacementof ACF by bovine pituitary extract (1 jtg/ml of culturemedium) is also found to be effective in restoring enzyme ac-tivity, directly confirming the autoradiographic experiments(Table II) and the observations presented in Figure 4. Con-centrations of pituitary extracts above 1 tg/ml of culturemedium cause a decrease in levels of enzyme activity. This ef-fect is related to a potent mitogenic effect of the pituitary ex-tract at that concentration, possibly due to an excess of fibro-blast growth factor (FGF). Under these conditions, cells pro-liferate and remain fusiform; both events are reminiscent of

those induced by exogenous prostaglandin F2. (PGF2.) whichbehaves as a potent mitogen (Negrel et al., 1981). Both ACFpurified from FCS and from the active fraction recoveredfrom bovine pituitary extract are dialyzable (mol. wt.<6000-8000), thermostable at neutral pH (10 min at 90°C),and are not inactivated by different enzymatic treatments(trypsin plus chymotrypsin, alkaline phosphatase, lipase plusesterase). Since untreated FCS when extensively dialyzed doesnot lose its activity for the formation of fat cell clusters, it ispossible that ACF is bound in serum to some protein com-ponent(s) and that dissociation of ACF occurs during ion-exchange chromatography or during heat treatment.

In an attempt to eliminate the possibility that ACF activitycould be attributed to some well characterized factor, adiposeconversion was examined on confluent Obl7 cells maintainedin resin-treated serum supplemented with insulin and T3, andby replacing ACF with the following substitutes: phospho-ethanolamine (0.5 -50 jiM) in the absence or in the presenceof prolactin (0.6-15 nM) (Kano-Sueoka and Errick, 1981),epidermal growth factor (1.7-17 nM), PGF2, (20-200 nM),17-a and 17-3 oestradiol (75 nM) (Roncari and Van, 1978),cortisol (0.2 ,uM), oestrone (75 nM), testosterone (70 nM),D-L-thyronine, 3,5-diiodo-L-thyronine, or thyroxine (TJ (2and 200 nM). All these components proved to have either noeffect or some inhibitory effect on adipose conversion. More-over, not only the mol. wt. ofACF (< 6000-8000) most like-ly excludes FGF and platelet-derived growth factor (PDGF),but also these mitogens were shown in separate experimentsto cause blockade of adipose conversion through continuousproliferation. Retention of ACF on strong anionic-exchangecolumn should also exclude polyamines (Bethell and Pegg,1981) and again PDGF. The apolar nature ofACF was exclu-ded, since full activity on adipose conversion was recovered inthe aqueous phase after treatment of purified ACF with amixture of solvents (Bligh and Dyer, 1959).

DiscussionWe have previously shown that T3 acts by amplifying the

expression of the specific phenotypes which emerge duringadipose conversion (Gharbi-Chihi et al., 1981). This hor-mone, active on a long-term basis at the cellular level, doesnot promote the formation of fat cell clusters. In contrast,ACF, partially purified from FCS, controls in some way thedevelopment of adipose clusters. It is clear that the amplifica-tion of adipose conversion after confluence by ACF is due toits specific mitogenic effect on susceptible cells. The lack ofACF effect on exponentially-growing cells thus may be ex-plained by the small proportion of susceptible cells presentduring the growth phase (P. Djian et al., in preparation)and/or by a specific requirement of susceptible cells in theresting phase or both.This specific requirement is supported by the data in Figure

6 which show that the continuous presence of ACF is not re-quired: a 3-day exposure of confluent cells to untreated serumis sufficient to obtain a full response if the cells are thenshifted to resin-treated serum for the remaining period of theresting phase. The chemical nature of ACF, which has beenrecovered in all batches of FCS treated so far, is presentlyunder investigation. It is of interest that bovine pituitary ex-tract, or a fraction recovered from this extract according tothe procedure used for ACF, can replace ACF for the forma-tion of fat cell clusters. Moreover, this active fraction andACF share similar chemical and biological properties, sug-

690

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Fig. 6. Minimal time of exposure to ACF for adipose conversion. 2 x 104cells were seeded and grown in 1007o resin-treated serum. At confluencethey were exposed for different periods of time, as indicated, to untreatedFCS and subsequendy shifted for the remaining period to resin-treatedserum. In each case assays were performed 13 days after confluence. 170nM insulin and 1.5 nM T3 were present throughout the resting phase.

gesting that ACF could originate from the pituitary. If it wereso, results of Figure 5 would indicate that ACF is - 500-foldmore concentrated in bovine pituitary extract than in FCS.

The relationship, if any, between ACF and the adipogenicfactor(s) characterized in crude extracts of bovine pituitary(Hayashi et al., 1981) or in FCS (Kari-Harcuch and Green,1978) remains to be shown since no purification of this fac-tor(s) has been reported so far. It could be envisioned thatACF plays the role of a specific mitogen, allowing post-con-fluent cells to become sensitive to the action of adipogenicfactor(s). The importance, if any, of this low mol. wt. com-ponent from serum in the control of adipose cell proliferationin vivo remains to be investigated in the mouse and otherspecies under pathophysiological situations leading to hyper-plasia of adipose tissue (Klyde and Hirsch, 1979).

Matenals and methodsCell culture

Unless otherwise stated, Obl7 cells were grown in Dulbecco's ModifiedEagle's Medium supplemented with 10%o FCS, 33 pM biotin, 17 PM panto-thenate, and antibiotics (Negrel et d., 1978). At confluence the following wereadded to the culture medium (changed every other day): 33 pM biotin, 17 PMpantothenate, 170 nM insulin, with or without 1.5 nM T3, in the presence of10%o untreated or treated FCS (see below) supplemented or not with ACF.Enumeration of cells was performed with a Coulter counter. Fat cell clusterswere stained with Oil RedO according to Green and Kehinde(1974). A fat cellcluster was defined as the smallest cluster visible after Oil Red 0 staining.Stainable dusters observed by microscopy were shown to contain at least60- 80 cells.Treatment ofFCS and bovine pituitary extracts with ACGX-10 resin

Treatment of FCS with AGIX-10 (Bidrad) was carried out according toSamuels et at. (1979). The serum, referred to as resin-treated serum, wasseparated from the resin by centrifugation. The resin was then washed for 15min at 20°C with 5 mM potassium phosphate buffer pH 7.2 (50 ml/g dryresin) and recovered by filtration on a BWichner filter. The active serum frac-tion (ACF) was eluted with 5mM potassium phosphate buffer pH 2.5 (2 h at20°C; 10 ml/g dry resin). After filtration the eluate was lyophilyzed. The dryresidue was dissolved in water and adjusted to 0.1 M potassium phosphate

buffer pH 7.0, sterilized by filtration using 0.2 Pm Millipore filters and storedat - 20°C. Control experiments showed that under these conditions labeledT3 and T4 (Gharbi-Chihi et at., 1981) remain bound to the resin and were noteluted. Bovine pituitary extract was obtained as described by Hayashi et at.(1981). The supernatant, called pituitary extract (105 000 g x 60 min; 5 mg/mlof protein), was treated with AGIX-10 (10 ml/g dry resin) and elution of anactive fraction was performed as described above. Heat treatment of activefractions was performed at 90°C for 10 min at pH 7.0. Trypsin alone (5000U/ml of active fraction) or in combination with chymotrypsin (25 U/ml) andpronase (90 U/ml) were used for proteolysis experiments. Active fractionswere also treated with alkaline phosphatase (1 U/ml) and with a mixture oflipase (50 U/ml) and esterase (1 U/ml).Enzyme assays

All enzyme assays were performed at least in duplicate on cell fractions oron cell homogenate from two pooled dishes (Grimaldi et al., 1978). Variationsbetween assays did not differ by more than 7% and variations between meanvalues (of homogenate or of cell fractions) from two separate dishes did notexceed 10%. Specific activities are expressed in nmol of productformed/min/mg of protein, except fatty acid synthetase (FAS) activity ex-pressed in nmol [14C]malonyl-CoA incorporated into fatty acids/min/mg ofprotcin. Proteins were estimated by the method of Lowry et al. (1951).Incorporation of [3H]thymidine and autoradiographs

Cells were maintained in the presence of 6 ACi [3H]thymidine for 48 h(specific radioactivity 3 Ci/mmol). Less than 1001o of the total radioactivitywas incorporated under these conditions. Cells were then washed twice withphosphate-buffered saline, treated with 5% trichloroacetic acid for 10 min at4°C and washed twice successively with 70%o ethanol, 95% ethanol, and abso-lute ethanol. Fixed cells were covered in the dish with emulsion (Kodak NTB2)previously warmed for 30 min at 400C. The autoradiographs were exposedfor 7-9 days and then processed (2 min with developer D19B Kodak, 10 swith 0.1% acetic acid and 10 min with fixative LX24 Kodak). Labeling indices(ratio of labeled cells to all cells) were estimated in clusters and outside clustersby counting 10 random fields (at least 2000 cells in each case). The number oflabeled cell dusters was measured on the total dish and the number of labelednuclei per cluster was determined by counting at least six clusters. A labeledcell cluster was defined arbitrarily as a cluster containing at least 20 labelednuclei and whose labeling index was at least 25%.Hormones and chemicals

T3 and T4 were purchased from Sigma. [1-14C]acetate was a product of the"Commissariat a l'Energie Atomique". [2-14C]Malonyl-CoA was purchasedfrom NEN Chemicals Gmbh. NADPH, malate dehydrogenase, and glucose-6-phosphate dehydrogenase were obtained from Boehringer Mannheim. Thesource of all other products has already been given (Gharbi-Chihi et at., 1981).

AcknowledgementsThe authors wish to thank Misses B. Wdziekonski and M. Cazales for experttechnical assistance and Mrs. G. Oillaux for skilful typing. Thanks are due toDr. E. Van Obberghen Schilling for stimulating discussion and carefulreading of the manuscript. This work was supported by grants from "CentreNational de la Recherche Scientifique" (ATP 7300-01), from "Commissariata l'Energie Atomique", from "Fondation pour la Recherche Meiicale", andfrom NATO (grant no. 1704).

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