THEJOURNALOF BIOLOGICAL CHEMISTRY
Vol. 258, No.6, Iwue of March 25, pp. 9559-3564, 1983
Printed in U.S.A.
Increased Synthesis and Accumulationof Phospholipids during
Differentiation of 3T3-Ll Cells into Adipocytes”
Ranganna Kasturi andSalih J. Wakilg
From the Verna and Marrs McLean Department
of Biochemistry, Baylor College of Medicine, Houston, Texas 77030
Conversion of 3T3-Ll preadipocytes to fully devel-
oped adipocytes incultureunder the influence of
dexamethasone, 1-methyl-3-isobutyl xanthine, and in-
sulin offers a unique system to investigate differenti-
ation-related changes in lipid metabolism. Depending
on the type of isotopic precursors @Hz]O, 32Pi,and [l-
14C]acetate) used,a 10-170-fold increase in the rate of
incorporation into lipid was observed in 3T3-Ll adipo-
cytes which contained 5-10-fold higher amounts of cel-
lular protein/culture than preadipocytes. In preadipo-
cytes and adipocytes, the major lipids synthesized were
phospholipids, triglycerides, and fatty acids. The rate
of 32Piincorporation into totallipids was 21-fold higher
in adipocytes than that in preadipocytes, and 90% of
the total radioactivity in both preadipocytes and adi-
pocytes was contributed by phosphatidylcholine (PC),
phosphatidylethanolamine (PE), and phosphatidylino-
sitol (PI). The content of phospholipids was ?-%fold
higher/culture in adipocytes than that in preadipo-
cytes, and 66 and 80% of the total phospholipids of
preadipocytes and adipocytes, respectively, were com-
posed of PC and PE. Based on DNA measurements,
there was a %fold increase in phospholipids and an
approximately $fold increase in protein/cell.
During 3T3-Ll adipose conversion, a detectable in-
crease in cellular protein, triglycerides, and phospho-
lipids was observed on day 3 after induction and, there-
after, these constituents increased continuously up to
day 11.Among the different phospholipids of both pre-
adipocytes and differentiating 3T3-Ll cells,PC, PE, and
PI exhibited significant changes in their rate of 32Pi
incorporation during adipogenesis. While there was a
continuous increase in the incorporation of 32Piinto PI,
there was preferential incorporation of 32Pi into PC and
PE, the significance of which is not clear. The increase
in phospholipids and protein content during adipo-
genesis suggests that phospholipids are required for
membrane biosynthesis.
3T3-Ll cells obtained by Green and Kehinde (1-3) fromthe
original stock of mouse embryo 3T3 cells undergo spontaneous
adipose conversion on reaching confluency. This spontaneous
adipose conversion is enhanced by exposing the confluent cells
to various hormones, drugs, and nutrients (1-5). During the
metamorphosis of highly extended preadipocytes to fully de-
* This work wassupported in part by Grants GM 19091, AM 21286,
and HL 17269 from the National Institutes of Health, Grant PCM 77-
00969 from the National Science Foundation, and Grant 6-587 from
The Robert A. Welch Foundation. The costs of publication of this
article were defrayed in part by the payment of page charges. This
article must therefore be hereby marked “aduertisement” in accord-
ance with 18 U.S.C. Section 1734 solely to indicate this fact.
$- To whom reprint requests should be addressed.
3559
(Received for publication, June 28, 1982)
veloped adipocytes, spectacular morphological and biochem-
ical changes occur. Along with the appearance of large lipid
droplets indifferentiating 3T3-LI cells, there is an increase in
the activities of various lipogenic enzymes involved in the
synthesis and accumulation of lipids (6-11) as well as lipolytic
enzymes that are engaged in mobilization of the stored lipid
(11, 12). In addition to an increase in enzyme activities, it is
also demonstrated that there is a dramatic change in protein
synthesis (13) and also in the amounts of proteins relating to
at least three distinct aspects of adipose conversion such as
triglyceride synthesis, glycolysis, and cell structure ( 14). 3T3-
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L1 cells acquire sensitiveness to hormones that are known to
affect adipose cells on differentiation (15-19).
Although it is well known now that 3T3-Ll cells differen-
tiate into fully developed adipocytes accompanied by morpho-
logical and enzymatic differentiation, sensitivity to hormones,
and accumulation of triglycerides, not much is known about
the other lipids synthesized by 3T3-Ll cells. Green and Ke-
hinde (1, 2) showed that only the amount of triglyceride
changes during differentiation. Hyman et al. (20) reported the
accumulation of (n-9)-eicosatrienoic acid in confluent and
differentiated 3T3-L1 cells.
In the present paper, we report that there is an increase in
the synthesis and content of phospholipids during adipose
conversion of 3T3-LI cells which may be required for mem-
brane biosynthesis.
EXPERIMENTALPROCEDURES
Culture Conditions-Culture dishes of 60 mm were seeded with 1-
2 X IO4 3T3-Ll cells and were grown to confluence in 3 ml of
Dulbecco’s modified Eagle’s medium supplemented with 10% fetal
calf serum, penicillin (50 microunits/ml), and streptomycin (50 pg/
ml) in a humidified atmosphere of 5% CO, and 95% air at 37 “C.
Cultures werefed every 2 days during growth and differentiation
unless otherwise mentioned. A day after confluence, the cells were
fed with Dulbecco’s modified Eagle’s medium containing 0.5 mM 1-
methyl-3-isobutyl xanthine and 0.25 PM dexamethasone to enhance
the differentiation process. The culture medium was removed after
72 h and replaced with Dulbecco’s modified Eagle’s medium contain-
ing 1.6 PM insulin. By 10-15 days after confluence, 8040% of the cells
in monolayer expressed the adipocyte phenotype. Undifferentiated
3T3-Ll cells were maintained in a similar condition except in the
absence of dexamethasone, I-methyl-3-isobutyl xanthine, and insulin.
Since the frequency of adipose conversion decreased on repeated
subculturing, the stock cultures of earlier passages (up to passage 5)
were used for inoculation.
Incorporation of [I-’4C]Acetate, (“HZIO, and ‘12Pm into Cellulur
Lipids of 3T3-Ll Cells-Cells were incubated in 2 ml of culture
medium containing 10 pCi of [l-I4CJacetate (57.7 mCi/mmol), I mCi
of 32Pl,or 2 mCi of [”H2]0for 1 or 2 h at 37 “C. The medium was then
withdrawn and the cells were washed three times with phosphate-
buffered saline, pH 7.4.
Extraction of Cellular Lipids-After washing, the cells were har-
vested by scraping with a rubber policeman and collected in 1 ml of
phosphate-buffered saline. Cells were sonicated three times at 10-s
intervals at 60 watts using a microtip (Heat Systems-Ultrasonics,
Inc., model W185). The cellular lipids were extracted with chloro-
3560 Increase in Phospholipids during3T3-Ll Adipose Conversion
form:methanol (2:l. v/v) (21).
Separation of Neutral Lipids-The extracted lipids wereseparated
by thinlayerchromatography onSilica Gel Gaccording tothe A
method of Skipski et al. (22) and Skipski and Barclay (23). The lipid
spots were detected either by exposing to iodine vapors or to x-ray
film when radiolabeled lipids were chromatographed.
Separation of Phospholipids-Phospholipids were separated by
two-dimensional thin layer chromatography on Silica Gel H plates
conta;ning 10% magnesium acetate, using ch1oroform:methanol:
ammonium hydroxide (28%) (65255.v/v)and ch1oroform:ace-
tone:methanol:aceticacid:water (304010101) as first and second PE
solvent systems, respectively. The spots were detected by exposing
theplatesto x-ray film. by staining with iodine, or by spraying
Phospray (Supelco, Inc., Bellefonte, PA).
Elution a n d Estimation of Different Phospholipids after Thin
Layer Chromatography-Individually separated phospholipids were
scraped off from the plate and elutedfrom the silica gel by following
the method described in Ref. 23. The lipid phosphorus content was
measured according to the procedureof Duck-Chong (24).
Extraction of Sterols and FattyAcids-Cells suspended in 1 ml of
phosphate-buffered saline weresaponified for 30 min in a boiling
water bath following the addition of 1 ml of 209 KOH and 1 ml of
ethanol. Nonsaponifiable sterols were extracted with pentane three
times with 3 ml each. Fatty acidswere extracted with pentane (3 X 3
ml) after acidification with 1 ml of 4 M H?SO,.
Extraction a n d Estimation of Triglycerides-Cells were collected

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in 0.5 mlof phosphate-buffered saline and sonicated as described
earlier. Triglycerides wereextracted from the sonicateusing activated
alumina and isopropyl alcohol. The triglycerides content was meas-
ured as described by Fletcher (25). The procedure involves an initial
step of saponification toliberate glycerol from triglycerides. The
released glycerol was oxidized to formaldehyde followed by quanti-
tation using Hantzsch's acetyl acetone color reagent. Triolein was
used as a standard.
DNA content was determined according toBurton's modified
method (26). Cellular protein content was measured by employing
the Lowry method (27). All the data presented in this paper were tI
obtained from at least two independent experiments and the samples
used were obtained by pooling four to six culture dishes. Variations
between independent experiments were within 15%.
I- ~~
"
~

FIG. 1. Phospholipids of 3T3-Ll preadipocytes and adipo-

RESULTS cytes. Lipids extracted from undifferentiated (A) and differentiated
( B )3T3-LI cells equivalent to one-half dish of cells were used for the
Cellular lipid synthesis was studied in preadipocytes and two-dimensional TLC separation of phospholipids. The solvent sys-
completelydifferentiated 3T3-Lladipocytes by incubating tems used were: 1) chloroform:methanol:ammonium hydroxide
the cells with [l-'4C]acetate or ["HrJO. As shown in Table I, (65:25:5) for the horizontal direction and 2) ch1oroform:acetone:meth-
anol:acetic acid:water (304010101) for the vertical direction. Spots
were identified by charing with 50% sulfuric acid. LPL, less polar
TABLE I lipids (cholesterol,triglycerides, etc.); W . X,X I . and Z, unknown,
Incorporation of [l-'4CJacetate a n d [JHJO into cellular lipids of nonphosphorus-containing spots;Sph, sphingomyelin; LPC, lyso-
3T3-LI preadipocytes a n d adipocytes phosphatidylcholine; PS, phosphatidylserine; PA, phosphatidic acid;
DPC, diphosphatidylglycerol.
After confluence, cells were treated with dexamethasone and 1-
methyl-3-isobutyl xanthine for 72 h and then fed with medium con-
taining 10 pg of insulin/ml for 12-13 days (about 90% of the cells in a 170-fold increase in the rate of incorporation of [1-i4C]
monolayerexpressed theadipocytephenotype).Undifferentiated acetate into lipids was observed in adipocytes. Furthermore,
3T3-Ll cells were maintained in a similar manner but in the absence
of dexamethasone, I-methyl-3-isobutyl xanthine,and insulin.Cul- the rate of synthesis of different lipids was also increased,
tures were pulsed with either [I-"C]acetate (10 pCi/dish) for 1 h or among which triglycerides (369-fold), phospholipids (148-
[:'Hz10 (2 mCi/dish) for 2 h. Lipids were extracted and separated by fold), fatty acids (118-fold), and cholesterol (88-fold) were
thin layer chromatography asdescribed under "Experimental Proce- prominent. However, both in preadipocytes and adipocytes,
dures." Triglyceride contents in the lipid extracts of preadipocytes the major lipids synthesized from[ l-i4C]acetatewere triglyc-
and adipocyteswere 0.056 and 2.72 mg/dish, respectively. The cellular erides and phospholipids.
protein content was 0.2 and 2.4 mg/dish in preadipocytes and adipo-
cytes, respectively. The values shown in parentheses represent the The increase in the rate of incorporation of [l-'4C]acetate
per cent total radioactivity found in each lipid. into lipids of 3T3-Ll adipocytes could result from a possible
difference in intracellular pool size of acetate or a difference
I [I-"CjAcetate [:'H2]0
Lipids in the uptake of acetate. Hence, a similar study was carried
Preadipo-
cytes Adipocytes prE$l.r- Adipocytes out using ["H2]0 as an alternateprecursor whose pool size is
large and does not vary significantly to affect the radiospecific
cpm/dish/h cpm/dish/h
Total 970
activity. The incorporation of [:'H2]0 into total cellular lipids
14,400 2,469,000 9.300
Triglycerides 3,514 (24) 1,296,000 (53) 263 (27) 6,733 (72) increased by about 10-fold in adipocytes. In comparison to
Phospholipids 4,910 (34) 718,000 (30) 390 (40) 1,134 (12) preadipocytes, there was about a 26-, 3-, 7-, and 4-fold increase
Fatty acids 2,577 (18) 306,000 (12) 158 (16) 1,050 (11) in the incorporation of [:'H2]0 into triglyceride, phospholipids,
Cholesterol 691 (5) 61,000 (2) 42 (4) 176 (2) fatty acids, and cholesterol, respectively, in adipocytes.
Diglycerides 2,131 (15) 54,000 (2) 95 (10) 158 (2) Studies on phospholipids were extended further to deter-
Monoglycerides 561 (4) 19.700 (1) 21(2) 56 (1) mine whether there are quantitative and qualitative differ-
 Increase in Phospholipids during3T3-Ll Adipose Conversion 3561
TABLEI1
Synthesis and content ofphospholipids in 3T3-Ll preadipocytes and adipocytes
Confluent cells weretreated with dexamethasone and 1-methyl-3-isobutyl xanthine for 72 h. Cells were then fed
with medium containing 10 pg of insulin/ml for 13 days. Undifferentiated 3T3-LI cells were maintained in an
identical condition but inthe absence of dexamethasone, I-methyl-3-isobutyl xanthine, and insulin. Cultures were
pulsed with 32Pi(1mCi/dish) for 1 h. Lipids were extracted and phospholipids were separatedby two-dimensional
thin layer chromatography and identified by exposing to x-ray fdm, by spraying with Phospray, or charing with
50% sulfuric acid as mentioned under “Experimental Procedures.” Lipid phosphorus content was measured as
describedunder“ExperimentalProcedures.” The valuesshowninparenthesesrepresent the percent total
radioactivity or per centtotal mass found in each individual phospholipid.
azp ,
, mcorporation Lipid phosphorus content
Phospholipids
Preadipocytes Adipocytes Preadipocytes Adipocytes
cpm/dish/h H
.~ / d i s h
Total 108,300 2,324,000 2.9 20.76
Phosphatidylcholine 56,973 (53) 1.34 (50)
1,154,917 (46) 10.82 (52)
Phosphatidylethanolamine 2,377 (2) 371,279 (16) 0.56 (19) 5.57 (27)
Phosphatidylinositol 38,477 (36) 603,113 (26) 0.27 (9) 0.77 (4)
Phosphatidylserine 2,599 (2) 39,268 (2) 0.05 (2) 0.24 (1)
Cardiolipin 297 (0.3) 46,980 (2) 0.07 (2) 0.44 (2)
Sphingomyelin 535 (0.5) 0.25 (0.2)5,665 (9) 1.49 (7)
Lysophosphatidylcholine 3,342 (3) 47,373 (2) 0.16 ( 6 ) 0.91 (4)
Phosphatidic acid 2,451 (2) 48,160 (2) 0.19 (7) 0.5 (2)
Phosphatidylglycerol 1,263 (1) 7,240 (0.3) NA“ NA
NA, not analyzed.

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ences between thephospholipids of undifferentiated and dif- TABLE111
ferentiated 3T3-Ll cells. The synthesis and contentof differ- Cellular DNA and lipidphosphorus content in 3T3-Ll
ent phospholipids as measured by the rateof incorporation of preadipocytes and adipocytes
32Pi and the amount of lipid phosphorus, respectively, were
Confluent cells were treated with dexamethasone and l-methyl-3-
elevated in adipocytesin comparison topreadipocytes as isobutyl xanthine for 72 h and then fed with medium containing 10
shown inFig. 1, A and B, as well as Table 11. Incorporation of pg of insulin/ml for12days.Undifferentiated3T3-LIcellswere
maintained inthe same way except in the absence of dexamethasone,
32Piinto cellular lipids increased by 21-fold in differentiated I-methyl-3-isobutyl xanthine, and insulin. The cellular DNA, protein,
3T3-Ll cells. There was abouta 156- and 158-fold increase in andlipidphosphorus content weremeasured as describedunder
the rate of incorporation of 32Piinto PE’ and cardiolipin, “Experimental Procedures.”
respectively. There was also about a 10-20-fold increase in the DNA Protein Lipid P, Idipid “/
incorporation of ”Pi into otherphospholipids (PC, 20-fold; PI, pg DNA
16-fold; phosphatidylserine, 15-fold; sphingomyelin, 11-fold; %/dish mg/dish &dish pg
lysophosphatidylcholine, 14-fold; and phosphatidic acid, 20- Preadipocytes 28.0 0.58 2.54 0.09
fold) of adipocytes. The 32Pincorporation intodifferent phos- Adipocytes 64.6 3.6 17.50 0.27
pholipids on a per cent basis was not very different in adipo-
cytes and preadipocytes (Table11). cytes, the time course of increase in synthesis and content of
The cellular lipidsof 3T3-Ll adipocytes contained about 7- these lipids during 3T3-Lladipose conversion was studied to
fold more phosphorus/culture than the corresponding 3T3-Ll determine the pattern of increase during the differentiation
preadipocytes (Table11).In addition, the phosphorus content process. In 3T3-Ll cells that were induced to differentiate, a
of each phospholipid of adipocytes was elevated in comparison 1.8-fold increase in the rateof 32P,incorporation was observed
to preadipocytes (Fig. 1,A and B, and Table11).There was a on days3 and 6 of adipose conversion (Fig.2 A ) . Subsequently,
3-10-fold increase in the phosphorus content of phospholipids the incorporation rate increased, and on day 11, a 9-fold
such as PC (8-fold), P E (10-fold), P I (%fold), phosphatidyl- increase in the ratewas observed. However, inundifferentiat-
serine (S-fold), cardiolipin(6-fold),sphingomyelin(&fold), ing preadipocytes, the rateof incorporation of :’‘Piinto cellular
lysophosphatidylcholine(6-fold), andphosphatidic acid (3- lipids remained almost constant throughout the experimental
fold). These results indicate that there is an increasein cellular period. The time course of increase in lipid phosphorus con-
lipids, particularly phospholipids and triglycerides on adipose tent during cell differentiation is illustrated in Fig. 2B. There
conversion of 3T3-Ll cells. was a rapid increase in lipid phosphorus content after 3 days
In all the above studies,adipogenesis was induced by treat- of differentiation indexamethasone-, 1-methyl-3-isobutyl xan-
ing dexamethasone- and 1-methyl-3-isobutyl xanthine-primedthine-, and insulin-treated 3T3-Ll cells and it remained con-
cells with 1.6 PM insulin. Since insulin is known tohave tinuously elevated throughout the experimental period. On
mitogenic effects, DNA measurements were made, both in day 11, there was about a 5.3-fold increase in lipidphosphorus
preadipocytes and adipocytes, to investigate whether the in- content of differentiating 3T3-U cells. The lipid phosphorus
crease in phospholipids on adipose conversion is due to an content of preadipocytes remainedfairly constant throughout
increase in cell number. As shown in Table 111, there was the experimental period. The data shownin Fig. 2C illustrate
about a 2.2- and 7-fold increase in DNA andlipid phosphorus, the timecourse of increase in adipocyte protein content both
respectively, in adipocytes. However, the lipid phosphorus/ in preadipocytes anddifferentiating 3T3-L1 cells. After day3,
pgof DNA increased by %fold, indicating that there is an there was a rapid increase in cellular protein content which
increase in phospholipids/cell on adipose conversion. continued to increase up to day 11, the longest time point
Since there was a major increase in membrane lipids and studied. On day 11, the adipocytes contained a 7-fold higher
storage lipids, ie. phospholipids and triglycerides, in adipo- amount of cellular protein/culture than preadipocytes, which
showed a negligible increase in protein during theexperimen-
’ The abbreviations used are: PE, phosphatidylethanolamine; PC, tal period. From our earlier studies, it was found that the
phosphatidylcholine; PI, phosphatidylinositol. increase in cellularprotein contentwas reflected in the signif-
3562 Increase
Phospholipids
in during
icant increase in microsomal proteins (5.9-fold) compared to
soluble proteins (data not shown).
The pattern of incorporation of 32Piinto different major
phospholipids during 3, 6, and 9 days of adipose conversion
was studied and compared with the pattern of corresponding
preadipocytes (Table IV). Among the different phospholipids
of both preadipocytes and differentiating cells, PC, PE, and
PI exhibited dramatic changes in their radioactivity with time.
In addition, on day 3, 6 , and 9 of the experimental period,
about 90-94 and 80-90% of the total radioactivity of preadi-
pocytes and differentiating cells, respectively, were comprised
only of three phospholipids, PC, PE, andPI. In preadipocytes,
the synthesis of PC was higher in the beginning of the exper-
ln-
e
3T3-Ll Adipose Conversion
imental period and declined thereafter. About 50% of the total
radioactivity was found in PC on day 3, while only 2% of the
total radioactivity was found in it on day 9. Similarly, in 3T3-
L1 cells that were induced to differentiate, there was higher
radioactivity in PC on day 3 which constituted about 60%of
the totalradioactivity. However, on day 6, the radioactivity in
PC delined to 30%of the totalradioactivity. This was followed
by an increase in radioactivity on day 9. On the other hand,
incorporation of"'Pi into PE of preadipocytes was low
throughout the experimental period and constituted about 1-
2%of the totalradioactivity. In contrast to preadipocytes, the
1.6
1.2
0.8
0.4
0
4

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0 3 6 9 1 ;?
DAYS AFlER CONFLUENCE
FIG. 2. Changes in phospholipids and cellular proteins as a
function of time. Changes in phospholipids as determined by "Pi
incorporation ( A )and lipid phosphorus content ( B )as well as cellular
proteins ( C )were followed as a function of time. Confluent cells (day
0) were used, and differentiation was induced by treating with dexa-
methasone and 1-methyl-3-isobutyl xanthine for 3 days (day 3) and
then with insulin up to day 11. On days 0, 3, 6,9, and 11, the control
(0)and treated (0)cultures were pulsed with "P, (1 mCi/dish) for 1
h. Lipids were extracted and radioactivity and phosphorus content
were determined. Total cellular protein content was measured after
the cells were sonicated.
DAYS AFTER CONFLWNCE
FIG. 3. Changes in triglycerides, fatty acids, and sterols as
a function of time. Changes in triglycerides at various times were
determined by estimating triglyceride content in the lipid extract ( A )
as well as byfollowing the rate of incorporation of [?H2]0 into
triglyceride ( B ) after separation by thin layer chromatography as
described under "Experimental Procedures." Changes in fatty acids
( C ) and sterols ( D ) as a function of time were monitored by the rate
of incorporation of ["Hz]O into saponifiable (fatty acids) and nonsa-
ponifiable (sterols) fractions of lipids. On days 0, 3, 6, 9, and 11, the
control (0)and theinduced (0) cells were pulsed with ["Hz10 (1mCi/
dish) for 2 h and incorporation into triglycerides, saponifiable and
nonsaponifiable fractions, was determined.

TABLEIV
Incorporation of "P,into phospholipids of 3T3-Ll cells during adiposeconversion
Confluent 3T3-Ll cells (day 0) were used.Dexamethasone and 1-methyl-3-isobutylxanthine treatment was given
for 72 h (3 days). Insulin treatment wasgiven after 72 h of dexamethasone and 1-methyl-3-isobutyl xanthine
treatment up to day 9. Undifferentiating 3T3-Ll cells were maintained in a similar condition but in the absence of
dexamethasone, 1-methyl-3-isobutyl xanthine, and insulin. Incorporation of "P, into lipids was determined by
pulsing cells for 1 h in the presence of 1 mCi/dish of 32P1.
Lipids were extracted and phospholipids were separated
as described under "Experimental Procedures," The datapresented are representative of duplicate determinations.
The values shown in parentheses represent the per cent total radioactivity found in each phospholipid fraction.
Days after confluency

Phospholipids 3 6 9

Control Induced Control Induced Control Induced

cpmldish cpmldish cpmldish
Total 70,735 130,585 74,255 139,450 84,550 404,685
Phosphatidylcholine 35,155 (50) 79,003 (60) 27,400 (37) 41,835 (30) 1,945 (2) 79,415 (20)
Phosphatidylethanolamine 1,061 (2) 12,405 (9) 1,262 (2) 33,468 (24) 761 (1) 77,699 (19)
Phosphatidylinositol 30,557 (43) 11,361 (9) 38,464 (52) 49,504 (35) 72,967 (86) 208,413 (52)
 Increase in Phospholipids
during
"Pi incorporation into PE increased with time of differentia-
tion of 3T3-Ll cells. The per cent of total radioactivity found
in PE of differentiating cells was 9, 24, and 19% on day 3, 6,
and 9, respectively. The time course of incorporation of 32P,
into PI was interesting bothin preadipocytes and differentiat-
ing 3T3-Ll cells. During the entire experimental period, the
rate of incorporation of 3zPiinto PI increased both in adipo-
cytes and preadipocytes. As shown in Table IV, 43-86 and 10-
52% of the total radioactivity were found in PI of preadipo-
cytes and adipocytes, respectively.
The time course of increase in triglyceride content and
synthesis of triglycerides and fatty acids as well as sterols
were studied during the process of adipose conversion (Fig. 3,
A-D). Both the triglycerides content and the synthesis, as
monitored by ['H2]0 incorporation, increased in differentiat-
ing cells with time, whereas, inpreadipocytes, the amountof
triglycerides and rate of [3Hz]0 incorporation into triglycer-
ides remained low and constant (Fig. 3,A andB ) .The increase
was obvious after day 3 of induction of differentiation. On day
11, a 38-fold increase in the rate of incorporation of ["Hz10
into triglycerides and a 15-fold increase in the amount of
triglycerides were observed in adipocytes. In differentiating
cells, the increase in incorporation of [3H2]0 into fatty acids
appeared to parallel the increase in incorporation of ['HHz]O
into sterols (Fig. 3, C and D). The incorporation of ['Hz10 into
fatty acids and sterols increased on day 3 and,thereafter, the
incorporation of the precursor continued toincrease through-
out theexperimental period. On day 11,the ratesof synthesis
of fatty acids and sterols in adipocytes increased by 40- and 9-
fold, respectively, in comparison to preadipocytes. Rate of
synthesis of fatty acids and sterols in preadipocytes did not
change significantly during the experimental period.
DISCUSSION
All previous investigations on 3T3-Ll adipocytes have em-
phasized the increase in the content andsynthesis of triglyc-
erides. The present study characterizes the changes in the
content and synthesis of phospholipids and triglycerides dur-
ing adipose conversion of 3T3-Ll cells. The rates of incorpo-
ration of [l-14C]acetate, ['HH~IO,and "Pi into phospholipids in
3T3-Ll adipocytes were 148-, 3-, and 21-fold, respectively,
higher than those in preadipocytes. The ratesof incorporation
of [l-I4C]acetate and ['H2]0 into triglycerides of 3T3-Ll adi-
pocytes were, respectively, 369- and 26-fold greater than those
in undifferentiated 3T3-Ll cells. In addition to theincrease in
the rateof 32Piincorporation into phospholipids of adipocytes,
there was a 7-fold increase in lipid phosphorus/culture or a 3-
fold increase/cell on adipose conversion. Furthermore, incor-
poration of "P, into individual phospholipids as well as the
phosphorus content of corresponding phospholipids also in-
creased on differentiation into adipocytes.
Green and Kehinde (1,2) induced adipose conversion in the
absence of exogenously added insulin, while Mack& et al. (6)
used insulin to accelerate adipose conversion. It is knownthat
insulin treatment increases the synthesis of proteins. Green
andKehinde (2) showed in stationary3T3-Ll adipocytes
maintained in the absence of insulin that there was no signif-
icantincrease in either cellular protein content or in the
incorporation of [l-14C]acetate into phospholipids. But in the
same report, theseauthors demonstrated that when insulin is
added to the culture medium, the protein content and the
incorporation of glucose into phospholipids in 3T3-U adipo-
cytes increased by 2- and 3-fold, respectively. A similar in-
crease in protein content was also obtained by Mackall et al,
(6) although there was no major incorporation of [1-l4C]ace-
tateinto phospholipids. On the contrary, using the same
isotope, we observed about a 148-fold increase in the incor-
3T3-Ll Adipose Conversion 3563
poration of [l-'4C]acetate into phospholipids constituting
about 30%of the total radioactivity. The major difference
between these studies is that in our studies we not only used
insulin to enhance adipogenesis, but we also primed the con-
fluent 3T3-Ll cells with dexamethasone and methylisobutyl
xanthine. However, Coleman et al. (28) observed about a 2-3-
fold increase in specific activities of choline and ethanolamine
phosphotransferases in 3T3-Ll differentiating cells in com-
parison to corresponding undifferentiated 3T3-Ll cells. Fur-
thermore, Grimaldi et al. (11) have demonstrated that the
incorporation of sn-[l-'4C]glycerol 3-phosphate into polar and
neutral lipids by cell-free homogenate of 3T3-Ll adipocytes
was 7.5- and 5.9-fold, respectively, higher than that intocon-
trol cell line 3T3-C2.
Concomitant with the increase in phospholipids, increase in
cellular protein content was observed in differentiated cells.
Depending on the ability of 3T3-Ll cells to differentiate, the
cellular protein content increased by about 5-lo-fold in com-
parison to preadipocytes. From our earlier studies, it was
found that the increase in cellular protein content was re-
flected in the significant increase in microsomal protein. How-
ever, the increase in phospholipids and increase in cellular
Downloaded from www.jbc.org by guest, on November 18, 2011
protein content during adipose conversion of 3T3-Ll suggest
that phospholipids are required for membrane biosynthesis
and also indicate that thereis increased membrane assembly/
cell.
Both in preadipocytes and adipocytes, 90-92% of the total
"Pi incorporation occurred in PC, PE, and PI. About 75 and
83% of the total lipid phosphorus of preadipocytes and adi-
pocytes, respectively, were formed of PC, PE, and PI. How-
ever, the rate of incorporation of 32P,into PI of both preadi-
pocytes and adipocytes is interesting. Although PI in preadi-
pocytes and adipocytes formed less than 10% (Table 11) of the
total lipid phosphorus, 26-85% (Tables I1 and IV) of '"Pi
incorporation into lipids of preadipocytes and adipocytes can
be accounted by PI. The rapid increase in the incorporation
of "Pi into PI compared to other phospholipids suggests a
rapid turnover of PI and a dynamic nature of PI pool. It has
been shown that a variety of hormones such as growth hor-
mone (29), thyrotropin (30), adrenaline (31, 32), vasopressin
(33,34),and insulin (35,36) increases the incorporation of :''Pi
into phospholipids of their target tissues. In many mammalian
tissues, addition of hormones or neurotransmitters that stim-
ulate calcium entry results in an increased breakdown of PI,
along with a secondary increase in the synthesis of this phos-
pholipid (37-39). It hasalso been shown to be involved in cell
proliferation (40,41) and in the attachmentof enzymes to the
plasma membranes (40). Stein and Hales(42) and De-Torron-
tegui and Berthet (36) found that insulin increased the incor-
poration of phosphate into rat fat-cell phospholipids and the
incorporation was more, particularly in PI. In our studies, the
adipose conversion of 3T3-Ll cells was induced by insulin
treatment of dexamethasone- and 1-methyl-3-isobutyl xan-
thine-primed cells. The specific increase in the incorporation
rate of 3'P, into PI during differentiation compared to other
phospholipids could be the effect of insulin. However, the
increase in "'Pi incorporation into PI of preadipocytes is dif-
ficult to explain on this basis, except if the small amount of
insulin present in the serum is sufficient to increase the
specific turnover of PI. However, for the time being, the
significance of increased turnover of PI both in preadipocytes
and adipocytes is not clear and it needs to be investigated
further.
Acknowledgment-We are grateful to Vasudev C. Joshi forhis
suggestions and thoughtful criticism throughout the course of this
study.
3564 Increase
Phospholipids
in during
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