Lipogenesis in primary cultures of adipoblasts derived from genetically obese Zucker rats

Lipogenesis

in Primary Cultures of Adipoblasts Genetically Obese Zucker Rats F. Bourgeois,

A. L. Goldstein,

Derived

From

and P. R. Johnson

Adipocyte precursor cultures prepared from the epididymal fat pads of genetically obese (fa/fa) and lean (Fa/Fa) Zucker rats grow similarly in culture. Addition of enriched medium (EM) containing human serum, insulin, and glucose stimulated lipid filling of the adipocyte precursors in both cultures. However, [“HI H,O incorporation into total lipids, fatty acid synthetase and lipoprotein lipase activities, and cytosolic protein contents are all decreased in the falfa compared with the Fa/Fa cultures. Substitution of lean or obese rat serum for human serum in the enriched medium does not alter the decreased lipogenic capacity of the fa/fa adipocyte precursor cultures.

I

T IS WELL KNOWN that obesity in humans tends to prevail in some families,’ in some ethnic groups’ and in some socioeconomic groups.3 Although there are compelling data from studies of twins and kinship groups that show that a significant “heritability factor” is present in human obesity,4 whether or not this factor may be attributable in some instances to a single gene, or is usually polygenic. is not known. However, a number of rodents inherit obesity through a single autosomal recessive gene. Such a single gene, which presumably results in a single lesion, is reasonably accessible to analysis at the cellular level, even though the final result of the presence of the genetic error is a complex metabolic disorder. Among the rodent strains that carry recessive genes for obesity, the genetically obese Zucker rat (fa/fa) has proved to be a reasonable animal mode1 for early onset human hypertrophichyperplastic obesity.5 The fa/fa rat has been shown to develop increased adiposity and increased adipose tissue lipoprotein lipase (LPL) as early as the first or second week of postnatal development.6 The obesity occurs in the absence of hyperphagia,6 hyperlipogenesis,’ hypertriglyceridemia,* or postnatal hyperinsulinemia.’ Furthermore, the obesity is not reversed by pharmacologic,” dietary,” or surgical’* intervention. In spite of the considerable data accumulated on obese Zucker rats, the mechanism of action of thefu gene remains unknown. One of the major problems inherent in investigating the primary locus of action of the gene is the inability to control or to account for the many in vivo factors that may be involved differentially as obesity develops in the rats. Thus, we have chosen the in vitro method of primary cell culture for our investigations of thefa gene, since this methodology allows the exploration odthe metabolism of populations of cells derived from a single tissue in an easily controlled milieu. Using such methodology we have previously established that the fu gene is associated with changes in cellular function of fetal hepatocytes.‘3”4 In addition,

Mefabdism, Vol.

32, No. 7 (July). 1983

we have partially characterized primary cultures of adipoblasts derived from the stromal vascular fraction of epididymal adipose tissue of fa/fa and Fa/Fa rats and have demonstrated that adipoblasts derived from both lean and fatty rats show similar patterns of growth under primary culture conditions and generally similar morphology.‘5 Conversion from nonlipid-filled precursor cell types to multilocular. and eventually unilocular adipocytes, occurs in both fatty and lean cultures after confluence and in the presence of enriched media. The adipoblast marker enzyme, lipoprotein lipase, is detectable in these cultures at six to seven days; however, both heparin- releasable and cell-associated LPL activity are depressed in fatty compared with lean cultures under all conditions studied thus far. This article is a report of additional studies of lipogenic metabolism in these adipoblast cultures. In the first set of experiments, [3H] Hz0 incorporation in total lipids and fatty acid synthetase (FAS) and LPL activities were measured after exposure of the established cultures to enriched media. In a second set of experiments, the above values were measured in adipoblast cultures exposed to enriched medium supplemented with either lean or fatty rat serum in order to ascertain whether or not in vivo “serum factors” affected in vitro adipoblast metabolism. From the Department of Biology, Vassar College, Poughkeepsie, NY. Received for publication February 3, 1983. Supported in part by NIH AMI 9382, AM 06197 and the March of Dimes Birth Defects Foundation. Address reprint request to Dr. P. R. Johnson, Box 377, Vassar College, Poughkeepsie, NY 12601. The present address for Dr. Bourgeois is INSERM IL Unite De Recherches sur la Nutrition et L’Alimenration, Hbpital Bichat. 170 bd Ney. 75877 Paris Cedex 18. The present address for Dr. Goldstein is Hoflmann-La Roche, Inc., Pharmacology II. Nutley. NJ 071 IO. 0 1983 by Grune & Stratton, Inc. 0026&049S/83~3207-0006$1.00/0

673

674

BOURGEOIS, GOLDSTEIN, AND JOHNSON

MATERIALS AND METHODS

Materials Types I and II collagenase were obtained from Worthington Biochemical Corp (Freehold, NJ); medium 199 H was obtained from Flow Laboratories (Rockville, MD); and heat-inactivated fetal bovine serum (FBS) was obtained from GIBCO (Grand Island, NY) and from KC Biological, Inc (Lenexa, KS). Purified insulin and cephalothin (Keflin) were generous gifts from Eli Lilly Research Laboratories (Indianapolis, IN). ScintiVerse reagent was obtained from Fisher Scientific Co (Springfield, NJ); Glycerol tri[9,10 (n) ‘Hloleate (approximately 600 Ci/mol) was obtained from Amersham Corp (Arlington Heights, IL); [2-“C]malonyl-CoA (15 mCi/mmol), [rH]H,O (100 mCi/g), and Liquifluor were obtained from New England Nuclear Corp (Boston, MA). Nylon mesh was purchased from Tetko, Inc (Elmsford, NY) and heparin from Calbiochem (La Jolla, CA). All other chemicals were supplied by Sigma Chemical Co (St Louis, MO).

Animals Homozygous lean (Fa/Fa) and fatty (fa/fa) male Zucker rats between IO and 12 weeks of age obtained from the Vassar College colony were the source of tissue for preparing cell cultures. Although fatty and lean animals at this age differ markedly in body weight, the number of adipocytes in their epididymal fat pads is the same.16 Rat serum was prepared from blood samples derived by aortic puncture from lean and obese male rats four to six months of age. All animals were routinely maintained in temperature controlled rooms with a 12-hour light/dark cycle before their use.

Media Growth medium (GM) consisted of Medium 199H buffered with 25 mmol/L bicarbonate and supplemented with 10% FBS (vol/vol) and cephalothin (0.1 mg/mL). Enriched medium (EM) was prepared by supplementing Medium 199H buffered with 25 mmol/L bicarbonate to pH 7.4 with insulin (8 x lo-’ mol/L), ghcose (11 mmol/L final concentration) and either 10% human serum (EM,) or 10% fatty (EM,) or lean (EM,) rat serum. The sera were sterilized by filtration through a 0.22 pm filter and stored at -70°C.

Cell Culture Adipoblast cultures were prepared using modifications of the method of Bjorntorp et al” and Glick and Rothblat’s as previously described.15

Assay Methods Lipoprotein lipase activity was measured by a modification of the method of Glick and Rothblat” as previously described.15 Fatty acid synthetase activity was measured according to the method of Mackall et alI9 One Milliunit of activity is defined as that which causes incorporation of 1 nmol/L malonyl CoA into fatty acids per minute. Before assay, FAS was released from the monolayers by the digitonin treatment method of Student et aLn’ The protein content of the cytosolic fraction produced by digitonin treatment was determined by the method of Lowry et al.” Total lipogenesis per monolayer was determined by [‘HI H,O incorporation into total Folch extractable lipid as previously described.” Incorporation of [3H] H,O into lipid classes was determined by thin-layer chromatography of Folch extracts. Aliquots of 100 rL were spotted on TLC plates pretreated with silica gel (Gelman Instrument Co., Ann Arbor, MI), and the plates were developed in a solvent system of heptane/diethyl ether/acetic acid (85:25:1). Areas containing phos-

pholipids, triglycerides, cholesterol, and free fatty acids were scraped into scintillation vials and counted in Liquifluor in a liquid scintillation spectrometer.

Experiment

1

Paired cultures of cells derived from fatty and lean rats were grown to confluence at six to seven days in growth medium. The growth medium was removed and EM” added to the monolayers. Fatty acid synthetase activity was measured at two, four, six, and eight days after the addition of EM,, using the combined cytosolic protein fraction released from two to six dishes. Heparin-releasable LPL activity was measured in duplicate plates. After two days in EM,, the medium was removed and replaced by 2 ml of sterile release medium (RM). Following two hours incubation, the RM was removed and replaced by EM”. This procedure was repeated on the same dishes after six days in EM”. Total lipogenesis was measured in duplicate or triplicate plates at days 2 and 6 after the addition of EM”. Data were expressed on the basis of cytosolic protein per dish.

Experiment

2

Cells derived from fatty and lean rats were grown to confluence in growth medium. The growth medium was removed and enriched medium supplemented with either human serum (EM,), fatty rat serum (EM,), or lean rat serum (EM,) was added. Determinations of LPL and FAS activities, and total lipogenesis after two and six days in the enriched medium were carried out as described above. The distribution of label into the various lipid classes was determined on Folch extracts from the six-day cultures.

Statistical Methods Two-way and three-way analysis of variance, the Neuman-Keuls test, and t tests were used. All data are reported as the mean k SEM.

RESULTS

Cell Culture Lean and fatty adipoblasts were plated at a density of 5 x lo5 ceils/60-mm dish in GM and reached confluence after six to eight days as previously described.” The growth medium was replaced by EM at confluence. Both lean and fatty adipoblasts began to accumulate lipid droplets after 24-36 hours in EM as previously described.15

Experiment

1

Total lipogenesis. Both genotype and time significantly affected [3H] H,O incorporation into total lipids (F = 43.764; df = 1; P 5 0.0001 and F = 13.205; df = 1; P I 0.003, respectively) (Fig. 1). More label was incorporated in lean than in fatty cultures, and the incorporation increased over time of exposure to the enriched medium. In addition, there was a significant interaction between genotype and time (F = 7.515; df = 1; P 5 0.02). Inspection of the data indicates that the incorporation of label by lean cultures showed a greater increase between days 2 and 6 in enriched media than did the fatty cultures, although post hoc analysis does not establish this point statistically.

LIPOGENESIS IN ZUCKER RAT ADIPOBLASTS

675

0

FoFo

m

fafo

0

FaFa

m

fafo

T

1 DAY5

6

DAYS

Fig. 1. [‘Hl)I,O incorporation of into total lipids in lean and fatty adipoblasts of Zucker rats. Each bar represents the average + SEM of duplicate or triplicate plates from four paired experiments. Open bar = Fa/Fa: solid bar = fa/fa.

Fatty acid synthetase activity. Both genotype and time significantly affected FAS activity (F = 87.064; df= l;P~O.OOlandF= 15.547;df=3;P10.0001, respectively) (Fig. 2). Activity was greater in lean than in fatty cultures and increased significantly over time of exposure to the enriched medium. In addition, there was a significant interaction between genotype and time (F = 6.267; df= 3; P 5 0.001). Activity increased significantly between days 2 and 4 and days 4 and 6 in lean cultures, while there was no significant increase over time in the fatty cultures.

1 DAYS

b

DAYS

Fig. 3. Heparin-releasable lipoprotein lipase activity in Zucher rat adipoblast after growth in enriched medium. Each bar represents the average LPL activity f SEM.

Lipoprotein lipase activity. Genotype significantly affected LPL activity (F = 6.330; df = 1; P I 0.03) with more activity in lean than in fatty cultures after exposure to the enriched medium (Fig. 3). There was no difference in LPL activity over time of exposure to the enriched medium. Sodium chloride inhibited LPL activity 90%-95% in both types of cultures. Protein content. The cytosolic protein content of lean adipoblast cultures was greater than that of fatty adipoblast cultures (1.6108 + 0.067 v 1.1925 * 0.027

0

FaFa

m

fafa

T

lT Fig. 2. Fatty acid synthetase activity in Zucker rat adipoblests after growth in enriched medium. Each bar represents the average FAS activity r SEM in adipoblast cultures from four paired experiments. Open bar = Fafa; solid bar = fafa.

‘/ 2 DAY5

L 4

DAYS

T

6

DAYS

8 DAYS

BOURGEOIS, GOLDSTEIN, AND JOHNSON

OH

uman serum

m

FaFa serum

a

fafa

serum

T

fafo

FaFa

6

DAY5

mg protein per monolayer; P c: 0.001) in enriched medium. Experiment

Fig. 4. Serum effects on [‘H]H,O incorporation into total lipids of Zucker rat adipoblasts. Each bar represents the average incorporation + SEM of duplicate or triplicate plates from four paired experiments. Open bar = EM,; solid bar = EM: Cross-hatched bar = EM,.

were supplemented did not influence the amount of label incorporated. Distribution of label among lipid classes. Genotype significantly affected the distribution of label among the various lipid classes (Table 1). Fatty adipoblasts incorporated a significantly greater percentage of label into phospholipids (F = 24.644; df = 1; P 5 0.001) and fatty acids (F = 12.916; df = 1; P 5 0.04) than did lean adipoblasts. Lean adipoblasts, however, incorporated a significantly higher percentage of label into the triglyceride fraction than did fatty cells (F = 10.960; df = 1; P 5 0.007). In addition, there was a significant effect of serum type on the amount of label incorporated into the fatty acid fraction. Inspection of the data suggests that the effect was the result of increased labeling of fatty acids in cells grown in

2

Total lipogenesis. Both genotype df= 1;P~0.0001)andtime(F=35.712;df=

(F = 30.494; ~;Ps 0.0001) significantly affected the amount of label incorporated into total lipids, while the serum type had no significant effect (F = 0.821; df = 2; P 5 0.448) (Fig. 4). Furthermore, there was a significant interaction between genotype and time (F = 6.908; df = 1; P 5 0.003). Lean cultures incorporated significantly more [3H] H,O into lipids than did fatty cultures after both two and six days of exposure to the enriched medium with a significant increase between days 2 and 6 as well. The source of serum with which the media

Table 1. Lipid Class Profile in Lean and Fatty Adipoblasts

after Six Days in Enriched Media

Percentageof RecoveredActiwty* Fa/Fa Lioid Class

PhospholipidS

EMut

2.3 + 0.4

EM.t

fa/fa EM.t

EM-t

2.5 + 0.1

2.9 r 0.2

7.3 + 1.4

EM, t

5.6 + 1.0 2.8 ? 0.0

Fatty Acid

1.6 + 0.2

1.6 + 0.2

2.1 + 0.1

1.7 * 0.2

2.0 + 0.2

Cholesterol

1.9 + 1.1

2.3 c 1.2

2.0 + 1.1

1.9 * 0.2

2.3 + 0.8

Triglyceride$

94.2

* 1.6

93.7

+ 1.1

93.1

+ 1.0

89.2

2 1.4

EM.t

5.1 + 1.3

90.5

r 1.6

1.7 + 0.4 89.8

f 1.4

Results reported are mean ? SEM of three experiments. *Percentage of recovered activity averaged 80% of initial activity par sample. tThere were no significant serum effects. tThe percentage of recovered activity in phospholipid was significantly greater in fa/fa than in Fa/Fa cultures. $The percentage of recovered activity in triglyceride was significantly greater in Fa/Fa than in fa/fa cultures. Note that 89%-94% was found in triglycerides.

of all recovered label

677

LIPOCENESIS IN ZUCKER RAT ADIPOBLASTS

OH m mfofo

uman FaFa

serum serum serum

fafa

FoFo 2 DAYS

6 DAYS

Serum effects on fatty acid synthetase activity in Zucker rat adipoblasts. Each bar represents the average FAS activity + SEM Fig. 5. in adipoblast cultures from four paired experiments. Open bar = EM”. . solid bar = EM,; cross-hatched bar = EM,.

medium containing fatty serum, but post hoc comparisons using the Neuman-Keuls test showed no significant effects between individual means. Fatty acid synthetase activity. Both genotype (F = 34.274; df = 1; P s 0.0001) and the time of exposure to enriched media (F = 64.041; df = 1; P 5 0.0001) significantly affected FAS activity, while the

type of serum with which the medium was supplemented did not (F = 0.424; df = 2; P 5 0.658) (Fig. 5). In addition, there was a significant interaction between genotype and the number of days of exposure to enriched media (F = 17.202; df = 1; P I 0.0001). Fatty acid synthetase activity was greater in lean than in fatty adipoblasts after both two and six days of

17

1s

0

Human serum

m

FaFa serum

a

fafo

14

Fig. 6. Serum effects on heparin-releasable fiirotein liise activity in Zucker rat adipobbsts. Each bar represents the average LPL acthritY f SEM of dupficate dishes from four paired experiments. Open bar = EM,: solid bar = EM,; cross-hatched bar = EM,.

3 1

1 -

serum

678

BOURGEOIS, GOLDSTEIN, AND JOHNSON Table 2. Serum Effects on Cytosolic Protein Content of Adipoblast Monolayers in Enriched Media* CytosolicProtein/Monolayert

(mg)

2 Days$ Group FaFa* faf.3.

EM,

l

l

6 Day& EM,

EM,

EM,

EM,

EM,

1.3874

+ 0.0528

1.3013

+ 0.1935

1.4383

f 0.2463

1.4495

r 0.0899

1.6295

+ 0.1952

1.8995

+ 0.1886

0.9865

+ 0.1424

0.9945

+ 0.2154

0.8948

+ 0.169 1

1.3468

I 0.1808

1.5818

+ 0.1003

1.4840

i 0.0877

*There is no significant serum effect on cytosolic protein. tThese data are the mean + SEM of duplicate determinations from 4-8 @IIs

paired experiments.

were maintained in EM for two days after confluence. Cytosolic protein content increased significantly over time in both Fa/Fa and fa/fa

monolayers. @ells were maintained in EM for six days after confluence. Cytosolic protein increased significantly over time in both Fa/Fa and fa/fa monolayers.

l*Fa/Fa

monolayers contained significantly more cytosolic protein than did fa/fa monolayers after both two and six days in EM.

exposure to the enriched medium. Post hoc analysis showed that while FAS activity increased significantly in lean cultures between two and six days in the enriched medium, (P 5 0.05) no significant change over time occurred in the fatty cultures. Lipoprotein lipase activity. Genotype significantly affected LPL activity (F = 35.334; df = 1; P 5 O.OOOl),with lean adipoblasts exhibiting greater activity than fatty adipoblasts (Fig. 6). Neither the number of days of exposure to the enriched medium (F = 0.011; df = 1; P s 0.917), nor the type of serum with which the medium was supplemented (F = 1S36; df = 2; P I 0.229) affected LPL activity. Protein content. Genotype (F = 10.058; df = 1; P 5 0.003) significantly affected the amount of protein assayed in the cytosolic fraction produced by digitonin treatment (Table 2). Lean monolayers contained more cytosolic protein than did the fatty monolayers under all conditions. In addition, the amount of protein increased with length of exposure to the enriched medium (F = 17.370; df = 1; P 5 0.0001). There were no significant effects related to serum type (F = 0.694; df = 2; P 5 0.506). DISCUSSION

Primary cultures of adipoblasts derived from the epididymal adipose tissue of 10 to 12-weeks-old obese Zucker rats exhibit depressed lipogenic activity when compared with cultures of similar cells derived from the adipose tissue of age-matched lean Zucker rats. Specifically, both heparin-releasable LPL activity and fatty acid synthetase activity are greater in lean compared with fatty adipoblasts after exposure to enriched medium, which enhances the lipid content of both types of cultures. Total lipogenesis as measured by [3H] H,O incorporation into extractable lipid is also depressed in fatty relative to lean adipoblasts, and parallels the difference seen in FAS activity. Ninety percent or more of the tritium label is recovered in the triglyceride fraction of the total lipid extracted from these cultures, but significantly more of the label is found in triglyceride in lean than in fatty adipoblasts. The adipoblasts derived from fatty rats have signifi-

cantly more label in the phospholipid and fatty acid fractions than do lean adipoblasts. This latter finding suggests that the fatty cultures may be synthesizing more membrane components than the lean cultures. Microscopic observation tends to confirm that more lipid accumulation occurs in the lean than in the fatty cultures after the addition of enriched medium. It is well established that the adipose tissue of obese Zucker rats is enlarged at lo-12 weeks of agel and that lipogenic activity in the tissue is elevated,22 as are the activities of both tissue LPLz3 and FAS.24 Thus the possible explanations for the differences seen in lipogenie activity in vitro and in vivo must be considered. One possible explanation is that the two cultures contain different populations of cells, since the stromal vascular fraction of the epididymal fat pads used to prepare the adipoblast cultures consists of a heterogenous cell population. If it is assumed that adipose tissue derived from 1O-l 2-week-old growing animals contains a fixed population of “precursor adipocytes” determined at some earlier developmental stage, it could then be argued that more of these precursors had differentiated and filled with lipid in the obese tissue than in the lean tissue, leaving fewer potential adipoblasts in the population of cells plated from the obese than from the lean tissue. However, this possibility is not supported by existing data. Johnson et alI6 have previously shown that adipose tissue from the epididyma1 pads of lo-12-week-old obese Zucker rats contains 3.5 x lo6 fat cells compared with 4.0 x lo6 cells for lean tissue. Also, they have established that the number of cells in the epididymal adipose depot continues to increase in both the obese and lean rats well beyond this time. ‘v’~Whether or not the increasing cell number represents true precursor proliferation or simply lipid-filling of already existing precursor cells remains an unresolved issue. However, there are a number of pieces of evidence that suggest that new adipoblasts may be formed in vivo throughout this period. In lipectomized Osborne-Mendel rats in which all precursors are removed, the cell number returns to normal values in the inguinal site over about a twomonth period of regeneration,25 since the tissue itself is

LIPOGENESIS

679

IN ZUCKER RAT ADIPOBLASTS

removed. In this instance, the new cells must come from newly differentiated precursors. Furthermore, morphologic evidence suggests that once precursors are determined, ie, acquire appropriate enzyme activity, they differentiate, ie, fill with lipid very rapidly.25 In addition, during the current primary culture studies, the two types of plated cells show identical growth curves, which also suggests, but does not establish beyond all doubt, that similar cell populations are present. In the Zucker rat fetal hepatocyte primary culture system, we have reported similar diminished lipogenic activity infu-gene bearing cultures compared with non-j&gene bearing cultures.” In this case when homogeneous populations of hepatic parenchymal cells are compared, it can be argued that the defect is of cellular origin per se and not a function of two differing cell populations. In the heterogenous population present in adipoblast cultures, the case is not so clear. Further studies must be carried out to quantitate the number of cells in each type of culture that do become lipid filled. Such studies are now underway in our laboratory. A second explanation is that the deficits seen in the cultures derived from fatty rats represent a cellular defect traceable to the mutant gene, which renders these cells incapable of maintaining even normal rates of anabolic activity under in vitro conditions. Such a defect might be compensated or even over-compensated by numerous stimulating factors in vivo. It seemed likely that serum would contain these stimulating factors in vivo26 and that, indeed, the finding of more “adipogenic” factors in the sera of fatty rats than in the sera of lean rats or humans might reasonably be expected. This hypothesis led us to expose the cultures to sera derived from fatty and lean rats. When fatty cultures were exposed to fatty serum, however, the deficit was not compensated, ie, their lipogenic activity was not enhanced by fatty serum. Thus, importantly, no evidence was found that an “adipogenic” factor is present in the sera of fatty Zucker rats in concentrations greater than that seen in the sera of either humans or lean rats. It remains the case that the cellular deficit expressed by fatty cells in vitro must respond to some combination of environmental stimuli, no doubt including serum factors, which results in the

enhanced lipogenic activity of both adipose tissue and liver in vivo. It may be that concentrations of triglyceride even greater than those provided by 10% serum supplementation of enriched medium will be necessary to provide adequate or hyperlipogenic activity by cultures derived from fatty rats. It is also possible that adipocyte-/stimulating serum fractions must be separated from adipocyte-/inhibiting fractions before serum effects can be detected in vitro.27 Another question raised by these in vitro findings is the possible nature of the cellular deficit. It has previously been reported that both fetal hepatocytes’4 and adipoblasts” bearing the f&gene show indications of depressed protein metabolism as well as depressed lipogenesis. Fetal hepatocytes incorporate less leucine into protein precipitates under conditions where intracellular leucine pools are similar, and adipoblasts derived from obese or lean rats are differently sensitive to the presence of penicillinstreptomycin in the culture medium. Since it is known that these antibiotics influence eucaryotic protein degradation,28 we have speculated that the differential sensitivity of the fatty adipoblasts may reflect altered regulation of protein degradation/turnover. The data reported in this study, ie, reduced activity of two lipogenic enzymes in fatty adipoblast cultures and the decreased cytosolic protein content of fatty cultures, are not inconsistent with our previous findings. In summary, the depressed lipogenic activity of primary cultures of adipoblasts derived from adipose tissue of young adult obese Zucker rats correlates with the similarly depressed lipogenic activity, depressed response to insulin, and depressed amino acid incorporation into protein previously observed in primary cultures of fetal hepatocytes bearing the fa gene. These metabolic deficits may reflect an underlying problem in a cellular regulatory mechanism, such as protein turnover, which may be traceable as a primary lesion produced by the fa gene. ACKNOWLEDGMENT We gratefully

acknowledge the expert technical assistance of Joyce E. Palmer.Dr. M.R.C.Greenwood assisted us in experimental design and Dr. Adena K. Student in discussions of methodology. We thank Ms. Evelyn Buzzy for expert preparation of the manuscript,

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BOURGEOIS, GOLDSTEIN, AND JOHNSON

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