Proc. Nati. Acad. Sci. USAVol. 77, No. 3, pp. 1321-1325, March 1980Biochemistry

Induction of pyruvate carboxylase apoenzyme and holoenzyme in3T3-L1 cells during differentiation

(3T3-L1 preadipocytes/biotin enzymes)

SVEND 0. FREYTAG AND MERTON F. UTTERDepartment of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106

Contributed by Merton F. Utter, December 5, 1979

ABSTRACT The specific activity of pyruvate carboxylase[pyruvate:carbon-dioxide ligase (ADP-forming); EC 6.4.1.11 in3T3-L1 cells increases approximately 20-fold when these cellsdifferentiate to an adipocyte-like form [Mackall, J. C. & Lane,M. D. (1977) Biochem. Biophys. Res. Commun. 79,720-725]. Aspecific antibody to the purified rat liver enzyme quantitativelyprecipitated pyruvate carboxylase from 3T3-L1 crude homog-enates. Use of this immunological technique permitted us todemonstrate that the increase in pyruvate carboxylase activityis due to an increase in the intracellular concentration of theenzyme. The content of pyruvate carboxylase in differentiated3T3-L1 cells is sufficienty high (1-2% of total protein) that theincrease in this large protein (subunit Mr = 130,000) can bevisualized when 3T3-L1 crude extracts are subjected to elec-trophoresis on sodium dodecyl sulfate/polyacrylamide gels.When 3T3-L1 cells differentiated in the presence of avidin, theycontained less than 5% of the pyruvate carboxylase activity ofcells that differentiated in the absence of avidin. However, theimmunoprecipitable pyruvate carboxylase content of the avi-din-treated cells was essentially the same as that of cells thatdifferentiated without avidin. Full activity of the enzyme wasrapidly restored in the avidin-treated celTs upon the additionof excess biotin. The recovery of activity was closely correlatedwith the incorporation of ['4Clbiotin into immunoprecipitablepyruvate carboxylase. The rapidity with which the activity wasrestored and the insensitivity of the process to inhibitors ofprotein synthesis strongly suggest that the apoenzyme of pyru-vate carboxylase accumulates during differentiation in thepresence of avidin.

Mouse fibroblasts of the 3T3-L1 line, under appropriate con-ditions, accumulate cytosolic triglyceride and acquire manyof the morphological and biochemical characteristics of maturemammalian adipocytes (1-4). This in vitro differentiationprocess is highly dependent upon hormonal stimulation byinsulin (3) and glucocorticoids (5). The accumulation of cyto-solic triglyceride is accompanied by markedly increased ac-tivities of many enzymes involved in triglyceride biosynthesis.Such enzymes include acetyl-CoA carboxylase (6), fatty acidsynthetase (6), ATP-citrate lyase (6), malic enzyme (7), glyc-erol-3-phosphate acyltransferase (4), pyruvate carboxylase (8),and others. Although it seems likely that the increases in theactivities of these enzymes represent increased intracellularcontent of the enzymes, this relationship has been establishedonly for acetyl-CoA carboxylase (6) and fatty acid synthetase(M. D. Lane, personal communication).

Pyruvate carboxylase [pyruvate:carbon-dioxide ligase(ADP-forming), EC 6.4.1.1] is found in significant amounts inanimals in liver, kidney, brain, and adipose tissue. In the lasttwo tissues it has been suggested that the enzyme plays a rolein fatty acid synthesis by participating in the processes oftransporting acetyl groups (as citrate) and reducing groups (asmalate) from the mitochondria to the cytosol. The marked in-

crease in the level of pyruvate carboxylase activity duringdifferentiation of 3T3-L1 cells to the adipocyte-like form isentirely consistent with such a role for the enzyme.

In this paper we report that the increase in pyruvate car-boxylase activity is due to an increase in the amount of the en-zyme as measured by immunoprecipitation. Cells that differ-entiated in the presence of avidin show low pyruvate carbox-ylase activity, raising the question of whether the apoenzymeis synthesized during differentiation of biotin-deficient 3T3-L1cells. Immunoprecipitation and rapid recovery of pyruvatecarboxylase activity upon addition of biotin indicate that apo-pyruvate carboxylase accumulated in such cells.

MATERIALS AND METHODSMaterials. Avidin (10.6 units/mg), bovine insulin, penicill-

in-G, Ca pantothenate, and streptomycin sulfate were obtainedfrom Sigma; d-[14C]biotin (55 Ci/mol; 1 Ci = 3.7 X 1010 bec-querels) was obtained from Amersham. Eagle's minimal es-sential medium with 4-fold increased concentrations of aminoacids and vitamins, in Earle's salts and without L-glutamine orNaHCO3, and fetal bovine serum were obtained fromGIBCO.

Cell Culture. 3T3-L1 cells obtained from the American TypeCulture Collection were grown in Eagle's minimal essentialmedium with 4-fold increased concentrations of amino acidsand vitamins, Earle's salts, 2 mM L-glutamine, 2.2 g of NaHCO3per liter, 8 ,ug of Ca pantothenate per ml, 100 units of penicillinper ml, 100 Aug of streptomycin per ml, and 10% fetal calf serum(complete medium). The cells were cultured in a 37°C hu-midified incubator with an atmosphere of 95% air/5% CO2.Monolayer cultures were plated in 60-mm (Corning) culturedishes at a cell density between 1.5 and 3.0 X 104 cells per cm2and the medium was changed every 2 days. When the culturesreached confluence (;7.5-10 X 104 cells per cm2) as deter-mined by phase-contrast microscopy, insulin was added to themedium at a final concentration of 10 /,g/ml. Where indicated,avidin was added to the medium at a final concentration of 0.1MuM and the avidin-containing medium was then incubated at37°C for 30 min before the medium was applied to the cul-tures.Enzymatic Assays. Cell monolayers were washed twice with

phosphate-buffered saline (4°C), then harvested in 1.0 ml of0.25 M sucrose containing 20mM 4-morpholinepropanesulfonicacid (Mops) (pH 7.4) and 3.0 mM EDTA by scraping with aplastic policeman. The harvested cells were frozen immediatelyin liquid nitrogen. To assay for enzymatic activities, we rapidlythawed the frozen cell suspension and disrupted the cells witha Brinkman Polytron apparatus at a medium speed setting for20 sec at 4°C. Pyruvate carboxylase was assayed by a H'4C03-fixation method in the presence of Triton X-100 according toAtkin et al. (9). For assay of malic enzyme, an aliquot of thecrude homogenate prepared as described above was centrifuged

1321

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1322 Biochemistry: Freytag and Utter

at 48,000 X g for 15 min and malic enzyme activity was mea-sured in the supernatant fraction according to Wise and Ball(10). All enzymatic activities were assayed at 370C. One unitof activity represents the formation of 1 Amol of product permin. Protein was determined by a modification of the Lowrymethod (11), with crystalline bovine serum albumin as thestandard.

Immunological Procedures. An aliquot of the crude ho-mogenate was frozen and thawed twice and centrifuged at48,000 X g for 15 min at 4VC. Pyruvate carboxylase was im-munoprecipitated from the supernatant fraction with antibodyagainst rat liver pyruvate carboxylase (12). The antigen-anti-body mixture contained 1% Triton X-405/20mM Na phosphateat pH 7.0 and was incubated at 370C for 30 min and then at4VC for 24 hr. The immunoprecipitates were collected bycentrifugation for 15 min at 19,000 X g and washed three timeswith the Na phosphate buffer containing 1% Triton X-405 and0.9% NaCl.

Determination of Amount of Pyruvate Carboxylase Pro-tein. The washed immunoprecipitate was solubilized in 30 yAof 1% NaDodSO4/1% 2-mercaptoethanol and placed in aboiling water bath for 10 min. The immunoprecipitates werethen subjected to electrophoresis on a NaDodSO4/7.5% poly-acrylamide slab gel (100 X 125 mm) with a 5% stacking gel andthe discontinuous buffer system of Laemmli (13). The gels werefixed with 15% trichloroacetic acid for 2 hr, stained in 0.25%Coomassie brilliant blue R-250 (prepared in 45% methanol/9.2% glacial acetic acid) for 3 hr, and destained in 33% meth-anol/10% acetic acid with gentle shaking. The gels werescanned at a wavelength of 570 nm with a Transidyne model2510 densitometer. The area under each peak was determinedwith the aid of an analog integrator.

Determination of [14C]Biotin in Pyruvate Carboxylase.The amount of [14C]biotin incorporated into pyruvate car-boxylase was determined by cutting a 2-mm slice containingthe pyruvate carboxylase band (see Fig. 3) from the gel andsolubilizing the radioactive material with 10 ml of a 3% Pro-tosol/Econofluor (New England Nuclear) mixture with shakingat 37°C for 48 hr. The radioactivity was then measured witha Packard Tri-Carb Liquid Scintillation Counter with the ef-ficiency determined by use of an internal standard.

RESULTSEffect of Avidin on Pyruvate Carboxylase Activity. The

specific activity of pyruvate carboxylase (milliunits/mg ofcellular protein) increased more than 20-fold in 3T3-L1 cellsmaintained for 16 days after confluence in the presence of amedium containing 10 ,ig of added insulin per ml. The specificactivity of pyruvate carboxylase in these differentiated cells(corrected for assay temperature) was comparable to that re-ported by Mackall and Lane (8). In contrast, 3T3-L1 cells al-lowed to differentiate in the presence of 0.1 ,uM avidin had onlyabout 5% of the pyruvate carboxylase activity of the control cellsat day 16 (Fig. 1). In addition, the specific activity of malicenzyme in the control cells at day 16 was 130 milliunits/mg incontrast to 30 milliunits/mg for the avidin-treated cells. Theseresults agree well with those of Kuri-Harcuch et al. (4), whofound the activity of malic enzyme to be reduced in biotin-deficient 3T3-L1 cells. By day 16, greater than 70% of the cellshad accumulated cytosolic lipid droplets to about the sameextent in both the avidin-treated and control cells. The amountof lipid accumulated by the avidin-treated cells varied fromexperiment to experiment and may be related to the fatty acidand lipoprotein content of the fetal bovine serum. To demon-strate that the low activity of pyruvate carboxylase was not dueto the presence of avidin or other inhibitors in the avidin-treated

4 6 8 11Days after

FIG. 1. Effect of avidin on pyruvate caiboxylase activity. 3T3-L1cells were allowed to differentiate in the absence (-) or presence (-)of 0.1 pM avidin. At the indicated days after confluence, duplicatedishes for each treatment were harvested and assayed for pyruvatecarboxylase activity. Each point represents the average of duplicatedeterminations.

cells, we added an aliquot of the homogenate from the avi-din-treated cells to an equal volume of a control homogenate.The mixture gave the expected average value for pyruvatecarboxylase activity. These results suggest that the low activityin the avidin-treated cells is due to biotin deficiency, with suchcells unable to synthesize the holoenzyme.

Increase in Amount of Pyruvate Carboxylase ProteinDuring Differentiation. Electrophoresis of 3T3-L1 crudeextracts on NaDodSO4/polyacrylamide slab gels disclosed thata protein, corresponding to pyruvate carboxylase in size(130,000 daltons), increased in amount during differentiation.Fig. 2 shows a slab gel to which was applied 25 ,ug of proteinfrom crude extracts obtained from cells at different days afterconfluence. Although the relative amounts of several proteinsincreased with time, the change in the protein correspondingto pyruvate carboxylase was the most dramatic. The amountof pyruvate carboxylase protein present appeared to be littleaffected by the presence of avidin.

0 2 4 6 9 12 1 5

66,000

. 45.000

s...4.34,700-24.000

FIG. 2. Relative increase in pyruvate carboxylase protein duringdifferentiation of :3T3-l1 cells. Protein from each crude homogenaterepresenting progressive days of differentiation in the absence (-)and presence (+) of avidin was subjected to electrophoresis. Thenumber above each track indicates the day after confluence. PC, 2 migof purified rat liver pyruvate carboxylase. Protein standards are bo-vine serum albumin (Mr 66,000), egg albumin (Mr 45,000), pepsin (Mr:34,700), and trypsinogen (Mr 24,000).

Proc. Natl. Acad. Sci. USA 77 (1980)

Proc. Natl. Acad. Sci. USA 77 (1980) 1323

Immunoprecipitation of Pyruvate Carboxylase and Cor-relation with Activity. If the increase in enzymatic activityobserved were due entirely to an increase in the enzymaticprotein, activity and specific protein should increase in parallel.To test this, we determined the amount of pyruvate carboxylaseprotein by quantitative immunoprecipitation of the 48,000 Xg supernatant fraction obtained by centrifuging cellular ho-mogenates. To validate this technique, it was necessary todemonstrate that (i) the 48,000 X g supernatant fraction con-tained all of the pyruvate carboxylase activity originally presentin the cell extract and (ii) this immunological procedure waseffective in immunoprecipitating all of the pyruvate carbox-ylase protein in the supernatant fraction. Disruption of the cellsby treatment with a Brinkman Polytron apparatus followed bytwo cycles of freezing and thawing showed that greater than95% of the pyruvate carboxylase activity in the original cellextract could be recovered in the 48,000 X g supernatantfraction. Sonification was also relatively effective in solubilizingpyruvate carboxylase activity, but homogenization was not.

Fig. 3 Upper shows the electrophoretogram of a Na-DodSO4/polyacrylamide gel of an immunoprecipitate obtainedwith antibody against rat liver pyruvate carboxylase from the48,000 X g supernatant fraction of differentiated 3T3-L1 cells.The migration of the pyruvate carboxylase band was identicalwith that of the purified rat liver enzyme. To demonstrate thespecificity of our antibody, we immunoprecipitated pyruvatecarboxylase from 3T3-L1 cells that had been incubated with[35S]methionine. Fig. 3 Lower shows that the antibody wasspecific for pyruvate carboxylase, with only a small amount ofother radioactivity present.

In order to quantitate the amount of pyruvate carboxylasein such immunoprecipitates, we constructed a standard curveby applying increasing amounts of the purified rat liver enzymeto slab gels. As shown in Fig. 4, there is a linear relationshipbetween the amount of purified enzyme applied to the gel and

I * -_%40PC HAB LAB

180 - Pyruvate carboxylase

160

140-

120

- I100 IEa 80

60 i HAB LAB

40

20

0 5 10 15 20 25 30 35Gel slice

FIG. 3. Immunoprecipitate of pyruvate carboxylase from 3T3-L1crude homogenate. Differentiated 3T3-L1 cells were incubated for48 hr with 2.5 1ACi of [35S]methionine per 5 ml in complete mediumcontaining 0.4 mM methionine and 10% fetal calf serum. Pyruvatecarboxylase was immunoprecipitated, washed, and subjected toelectrophoresis. The gel was stained for protein (Upper); one trackof the slab gel was cut into 352-mm slices and each slice was analyzedtor 35S (Lower). H AB, heavy-chain immunoglobulin; L AB, light-chain immunoglobulin; PC, pyruvate carboxylase.

Purified rat liverpyruvate carboxylase, ,g

FIG. 4. Standard curve of purified rat liver pyruvate carboxylase.Various amounts of purified enzyme were subjected to electrophoresisin NaDodSO4/polyacrylamide slab gels, fixed, stained, destained, andscanned at 570 nm with a densitometer equipped with an analog in-tegrator. The area under each pyruvate carboxylase peak is expressedrelative to the area under the peak corresponding to 8 Atg of purifiedprotein. Each point represents the average of duplicate determina-tions; bars represent the range.

the area under the peak in the densitometric scan (corre-sponding to pyruvate carboxylase) over the range 1-8 jig. Astandard curve of this type was run with each slab gel and usedto calculate the amount of pyruvate carboxylase in the immu-noprecipitates analyzed on the same gel. *To demonstrate that antibody against rat liver pyruvate

carboxylase could precipitate all of the 3T3-L1 antigen in the48,000 X g supernatant fraction, we performed two experi-ments. Fig. 5 shows a precipitin curve in which increasingamounts of antibody were added to a constant amount of extractcontaining pyruvate carboxylase. The amount of pyruvatecarboxylase precipitated was measured by the gel techniqueas described above. The results show that 25 milliunits of ac-tivity could be precipitated by 1.5 mg of antibody. At this point,the supernatant fraction contained no detectable pyruvatecarboxylase activity after centrifugation of the immunopreci-pitate. As additional evidence that the immunoprecipitationprocedure completely precipitated the enzyme, pyruvatecarboxylase was immunoprecipitated from a 3T3-L1 extractfrom cells that had been incubated with [35S]methionine asdescribed in the legend of Fig. 3. After centrifugation of theimmunoprecipitate, an aliquot of an unlabeled 3T3-L1 extractcontaining 25 milliunits of pyruvate carboxylase activity wasadded to the supernatant fraction. An additional amount ofantibody was added to this mixture (1.5 mg); then pyruvatecarboxylase was precipitated a second time and the precipitatewas subjected to gel electrophoresis. The pyruvate carboxylaseband contained no radioactivity, indicating that the labeledantigen had been completely precipitated with the first im-munoprecipitation.

* This procedure assumes that the binding of Coomassie blue is iden-tical for pyruvate carboxylase from rat liver and 3T3-L1 cells. Theenzymes appear to be very similar immunologically. However, iftheir dye-binding properties are not identical, the calculations madein this report (see Table 1) would be valid in a relative sense.

Biochemistry: Freytag and Utter

1324 Biochemistry: Freytag and Utter

1.00

0.8o3 m

.'

E a 0.6-'

aW W

. 0.4

a: 0.2

0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 2.2Antibody added, mg

FIG. 5. Immunoprecipitin curve of 3T3-L1 pyruvate carboxylase(PC). Pyruvate carboxylase was immunoprecipitated from crudehomogenates with increasing amounts of antibody against rat liverpyruvate carboxylase and a constant amount of antigen (25 milli-units). The immunoprecipitates were subjected to electrophoresis,and the amount of pyruvate carboxylase in each immunoprecipitatewas determined from a standard curve of the purified rat liver enzymeas described in Fig. 4. The amount of antigen precipitated is expressedrelative to that immunoprecipitated (3.8 ,ug) with 1.5 mg of antibody.Each point represents the average of triplicate determinations.

By the immunological methods described above, the amountof pyruvate carboxylase in 3T3-L1 cells was determined atvarious times during differentiation. Because the amount ofactivity varied over a 20-fold range, it was necessary to vary theamount of antibody used. In cells allowed to differentiate in theabsence of avidin, this was accomplished by measuring thepyruvate carboxylase activity and calculating the amount ofantibody needed on the basis of the results in Fig. 5, in which1.5 mg of antibody precipitated pyruvate carboxylase equiv-alent to 25 milliunits of activity. For cells grown in the presence

of avidin, where apoenzyme was present, the amount of anti-body used was adjusted on the basis of The enzymatic activityof the corresponding control cells.

Table 1 shows the amount of immunoprecipitable pyruvatecarboxylase in cells at different times during differentiation inthe absence and presence of avidin. The amount of pyruvatecarboxylase activity and the amount of enzymatic protein in-creased steadily from day 6 after confluence until about day16. At this point, pyruvate carboxylase protein represented1.76% of the total cellular protein. The specific activity of py-

Table 1. Relationship between pyruvate carboxylase activity andspecific immunoprecipitable protein during differentiation in the

presence and absence of avidin

Totalactivity/

Immuno- immuno-Enzyme precipitable precipitableactivity, enzyme, enzyme,*

milliunits/mg '4g/mg milliunits/,4gDay protein protein enzyme

Without avidin6 38.9 + 3.4 6.2 0.4 6.3 0.59 82.1 + 7.7 13.3 + 1.0 6.2 + 0.312 94.1 0.9 19.4 + 0.6 4.9 0.215 114.1 i 2.0 17.0 + 0.7 6.7 + 0.416 119.1 + 9.4 17.6 0.6 6.8 0.6

With avidin6 3.3 + 0.2 5.0 + 0.2 0.67 + 0.39 4.8 0.4 11.1 + 1.2 0.43 + 0.6

12 6.4 + 0.9 16.1 + 2.1 0.40 + 0.715 5.8 + 0.3 16.5 + 1.6 0.35 + 0.516 6.2+0.1 17.1 +0.4 0.36+0.1

One milliunit represents 1 nmol of oxalacetate formed per min.* Calculated by dividing column 2 by column 3.

ruvate carboxylase, obtained by dividing the total activity bythe amount of immunoprecipitable protein, was essentiallyconstant throughout differentiation. The lower half of Table1 shows that although there was little increase in pyruvatecarboxylase activity in cells treated with avidin, the increasein immunoprecipitable pyruvate carboxylase paralleled theincrease in the control cells quite closely. The calculated specificactivity of the pyruvate carboxylase in such cells is only about5% of that of the control cells. These results are consistent withthe conclusion that synthesis of the apoenzyme of pyruvatecarboxylase occurs in the absence of biotin.

Recovery of Pyruvate Carboxylase Activity in Biotin-Deficient Cultures. If the biotin-deficient cells contain thenative apoenzyme of pyruvate carboxylase, the activity shouldbe restored upon addition of biotin. Fig. 6 demonstrates thatmost of the expected activity was recovered within 4 hr afteraddition of biotin and that the activity after 12 hr was equal tothat of the control cells (see Table 1). Inclusion of 10,M cy-

cloheximide in the culture medium had no effect on the re-

covery of the activity over the first 10-12 hr (Fig. 6) althoughthe same concentration of cycloheximide decreased the in-corporation of [3H]leucine into trichloroacetic acid-precipitableprotein by 70% during the same period (data not shown). Bio-tin-deficient cultures that were incubated without added biotinshowed no increase in pyruvate carboxylase activity during theexperimental period. In addition, Fig. 6 shows that the incor-

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FIGI. 6. Recovery of pyruvate carboxylase (PC) activity in avi-din-treated cells. On day 16 after confluency, 5 ml of complete culturemedium containing 2 ,Ci of [l4Clbiotin (55 Ci/mol; final concentra-tion, 7.8 pM) was added to each of 16 dishes of the avidin-treatedcultures. To half of these dishes, cycloheximide (final concentration,10 ,M) was added 1 hr before addition of biotin. At the indicatedtimes thereafter, dishes for each treatment were harvested and as-sayed for pyruvate carboxylase activity. Then pyruvate carboxylasewas immunoprecipitated from the clarified crude homogenate. Theimmunoprecipitates were subjected to electrophoresis, and theamount of pyruvate carboxylase in each immunoprecipitate was de-termined. The pyruvate carboxylase band was cut out from the gel,and the radioactive material was solubilized and measured. Resultingspecific activity in cultures not treated with cycloheximide in thepresence (0) and absence (0) of added biotin. Specific activity incultures treated with cycloheximide in the presence (*) and absence(0) of added biotin. X, I'4ClBiotin incorporated into pyruvate car-boxylase in the absence of cycloheximide. Each point represents theaverage of triplicate determinations. (Inset) Autoradiogram of[14C] biotin-labeled immunoprecipitates and crude homogenates.['4C]Biotin-labeled immunoprecipitates (lanes 1-:3, done in triplicate)and crude homogenates (lanes 4 and 5, in duplicate) representing 24hr after addition of f'4C]biotin were subjected to electrophoresis ina NaDodSO4/7.5% polyacrylamide slab gel. The gel was dried andexposed to Kodak x-ray film SB-5 for 1 week; then the film was de-veloped.

> '123.45

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Proc. Natl. Acad. Sci. USA 77 (1980)

Proc. Natl. Acad. Sci. USA 77 (1980) 1325

poration of ['4Cjbiotin into immunoprecipitable pyruvatecarboxylase closely paralleled the restoration of activity. Fig.6 Inset shows an autoradiogram of a slab gel in which pyruvatecarboxylase was immunoprecipitated from cells 24 hr after theaddition of ['4C]biotin as well as the 24-hr biotin-labeled crudehomogenate. Pyruvate carboxylase was the only biotin-labeledprotein observed in this experiment. Acetyl-CoA carboxylase(Mr 240,000) is present in 3T3-L1 cells (6), and its failure toappear in the autoradiogram may indicate that little of theapoacetyl-CoA carboxylase was present at the time of biotinaddition. Other biotin-containing enzymes usually found inmammalian cells, such as propionyl-CoA carboxylase andf3-crotonyl-CoA carboxylase, may be present in amounts toosmall for detection.

DISCUSSIONThe short-term regulation of pyruvate carboxylase activity inanimal tissues can occur through changes in the concentrationsof substrate, particularly pyruvate, through inhibition by ADP(14), a product of the reaction, or through acetyl-CoA, a positiveeffector (15). The available evidence suggests that this enzymedoes not undergo phosphorylation, at least in rat liver (16). Morerecent evidence indicates that long-term regulation in rat livermay occur through changes in the intracellular concentrationof the enzyme. The amount of hepatic pyruvate carboxylaseprotein in rats may vary up to 3-fold, depending on the statusof the thyroid hormones (13). Also, the amount of enzymaticprotein doubles in experimental diabetes (M. B. Weinberg andM. F. Utter, unpublished results). In both instances, the changesin enzymatic content appear to be mediated through changesin the rate of synthesis.

Pyruvate carboxylase in 3T3-L1 cells is also regulated bychanges in intracellular concentration. The change in enzymaticcontent may be under hormonal control although it is not clearwhether the increase in synthesis of pyruvate carboxylase isdirectly controlled by the lipogenic hormone (insulin) or

whether the increase reflects the general differentiation process.

Preliminary experiments also suggest that addition of cyclicAMP to the cultures after differentiation has occurred causes

a marked decrease in pyruvate carboxylase activity. Revers-ibility of enzymatic content upon withdrawal of the hormoneshas not been documented.The data presented here, along with those of Coleman et al.

(17), suggest that the induction of pyruvate carboxylase maynot be dependent on de novo fatty acid synthesis. In avidin-treated 3T3-L1 cells, the synthesis of palmitate from acetateis severely inhibited (17) and under similar biotin-deficientconditions some of the lipogenic enzymes, such as malic enzymeand glycerol-3-phosphate acyltransferase (4), are not formed.Upon the addition of biotin, these enzymatic activities and fattyacid synthesis from acetate increase to control levels in 48 hr.

The time required for fatty acid synthesis to return to controllevels (8 hr) agrees well with the recovery of pyruvate carbox-ylase activity reported in this paper. As noted earlier, malicenzyme did not increase significantly in activity in the avi-din-treated cells in the present experiments. In contrast, theapoenzyme of pyruvate carboxylase was synthesized at the samerate as the holoenzyme in the control cells (Table 1). These re-sults suggest that the synthesis of the apoenzyme is not depen-dent on the presence of intermediates of fatty acid synthesis.The avidin-treated 3T3-L1 cell appears to be a very useful

system for studying the relationships between the apoenzymeand holoenzyme of pyruvate carboxylase. The amounts of theenzyme are relatively high and the turnover is reasonably rapid(22- to 28-hr half-life, unpublished results). Thus, the systemshould provide an opportunity to compare the regulation of theturnover of the apoenzyme and holoenzyme and their inter-conversions.

We thank Drs. Richard E. Miller and Howard Gershman for theirassistance and advice on the cell culturing techniques and for theirhelpful discussions of the experiments described here. This researchwas supported by Grants AM-12245 and 5T32 GM-07225 from theNational Institutes of Health.

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53-59.5. Rubin, C. S., Hirsch, A., Fung, C. & Rosen, 0. M. (1978) J. Biol.

Chem. 253, 7570-7578.6. Mackall, J. C., Student, A. K., Polakis, S. E. & Lane, M. D. (1976)

J. Biol. Chem. 251, 6462-6464.7. Kuri-Harcuch, W..& Green, H. (1977) J. Biol. Chem. 252,

2158-2160.8. Mackall, J. C. & Lane, M. D. (1977) Biochem. Biophys. Res.

Commun. 79, 720-725.9. Atkin, B. M., Utter, M. F. & Weinberg, M. B. (1979) Pediatr. Res.

13,38-43.10. Wise, E. M. & Ball, E. G. (1964) Proc. Natl. Acad. Sci. USA 52,

i255-1263.11. Hartree, E. F. (1972) Anal. Biochem. 48,422-427.12. Weinberg, M. B. & Utter, M. F. (1979) J. Biol. Chem. 254,

9492-9499.13. Laemmli, U. K. (1970) Nature (London) 227,680-685.14. Walter, P. (1976) in Cluconeogenesis, eds. Hanson, R. W. &

Mehlman, M. A. (Wiley, New York), pp. 239-265.15. Barritt, G. J., Zander, G. L. & Utter, M. F. (1976) in Gluconeo-

genes*s, eds. Hanson, R. W. & Mehlman, M. A. (Wiley, NewYork), pp. 3-46.

16. Leiter, A. B., Weinberg, M. B., Isohashi, I. & Utter, M. F. (1978)J. Biol. Chem. 253,2716-2723.

17. Coleman, R. A., Reed, B. C., Mackall, J. C., Student, A. K., Lane,M. D. & Bell, R. M. (1978) J. Biol. Chem. 253,7256-7261.

Biochemistry: Freytag and Utter