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Changes in Angiotensinogen Messenger RNA
in Differentiating 3T3-F442A Adipocytes
JoAnne Saye, Kevin R. Lynch, and Michael J. Peach
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Angiotensinogen messenger RNA (mRNA) has been identified in both brown and white adipose
tissue. Recently we have shown that when 3T3-L1 cells were treated with isobutylmethylxanthine (IBMX) to accelerate differentiation, angiotensinogen mRNA increased markedly in
adipocytes as compared with preadipocytes. To determine if a correlation existed between the
regulatory events associated with the differentiation process, we compared the change in
angiotensinogen mRNA in spontaneously differentiating 3T3-F442A cells with two established
parameters of differentiation in adipocyte cell lines. Differentiation was assessed by visual
examination of cells for lipid droplets, fluorescent staining of the F-actin fibers, and increases
in glycerol phosphate dehydrogenase mRNA. F-actin fibers were highly structured in preadipocytes, becoming disassembled and very disorganized as cells differentiated into adipocytes.
The quantity of angiotensinogen mRNA increased as the number of lipid-containing cells
increased within a culture. Glycerol phosphate dehydrogenase mRNA accumulated in differentiated adipocytes to about the same extent as angiotensinogen mRNA. Thus, increases in
angiotensinogen mRNA were associated with the morphological and biochemical changes that
occur during the phenotypic modulation of 3T3-F442A cells. {Hypertension 1990;15:867-871)
A ngiotensinogen is the precursor to angiotensin
/ \
II, which is important in maintaining fluid
.Z \ . and electrolyte homeostasis and blood pressure. Angiotensinogen messenger RNA (mRNA) has
been found in brown and white adipose tissue, fibroblastlike cells of the periaortic connective tissue,1-3
and in the model fibroblast-adipocyte system, 3T3-L1
cells. 4 Angiotensinogen mRNA was markedly
increased in the fully differentiated adipocytes when
differentiation was accelerated and synchronized by
treatment with dexamethasone and isobutylmethylxanthine (IBMX) but not in untreated 3T3-C2 fibroblasts. This result suggested that the regulatory events
controlling differentiation of the LI cells also regulated angiotensinogen gene expression.4 When C2
cells were subjected to the same regimen (steroid+
IBMX), however, angiotensinogen mRNA was
detected and increased in the same time-dependent
manner as seen in the LI cells although not to the
same extent. The present study was done to determine
if phenotypic modulation played a role in the expression of the angiotensinogen gene in 3T3-F442A cells
Methods
3T3-F442A cells (provided by H. Green, Harvard
Medical School, Cambridge, Massachusetts) and control 3T3-C2 cells, a clonal line with a low frequency of
differentiation into adipocytes, were cultured as previously described.4 For experiments, 3T3-F442A cells
were grown to confluence and allowed to differentiate
spontaneously without the addition of drugs. On confluence, flasks were examined every 1-2 days with a
phase-contrast microscope to assess the approximate
percentage of lipid-containing cells. At no time were
lipid droplets observed in the time-matched C2 cells.
Complete differentiation of the F442A clones occurred
in 28-35 days.
From the Department of Pharmacology, University of Virginia
School of Medicine, Charlottesville, Virginia.
Supported in part by grants P01-HL-19242 (MJ.P.) and RO133513 (K.R.L.) from the National Heart, Lung, and Blood Institute. K.R.L. is an Established Investigator for the American Heart
Association with partial funding provided by the Virginia Affiliate.
Address for correspondence: Michael J. Peach, PhD, Department of Pharmacology, University of Virginia School of Medicine,
Box 448, Jordan Hall, Charlottesville, VA 22908.
Total RNA was extracted from the cells of one 75
cm2 flask of both cell lines as the percentage of lipidcontaining F442A cells increased; RNA was measured
spectrophotometrically (Gilford Instruments, Oberlin,
Ohio).2 Total RNA was analyzed either by Northern
gels or dot blots; angiotensinogen mRNA was detected
with the hybridization of a radiolabeled full-length
complementary DNA (cDNA) for rat angiotensinogen3
that had spontaneously differentiated into adipocytes
in the absence of drugs. Angiotensinogen mRNA
accumulation was compared with accumulation of
glycerol phosphate dehydrogenase (GPD) mRNA, an
established marker of differentiation in adipocytes,5-7
and with the change in the intermediate actin fibers
that change with differentiation.8
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Vol 15, No 6, Part 2, June 1990
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II
and GPD with a cDNA provided by B. Spiegelman
(Harvard Medical School).5 Blots were probed with
angiotensinogen cDNA, exposed to film, then washed
extensively with xO.l SSPE, and probed with GPD
cDNA.
Cells were grown in Falcon (Fisher Scientific,
Pittsburgh, Pennsylvania) 35-mm culture plates and
stained for F-actin (intermediate actin) with
I
1
rhodamine-phallocidin, a fluorescent probe, by the
method of Borak et al.9
Results
F442A cells were allowed to differentiate spontaneously in the absence of exogenous drugs; C2 cells
were maintained in the same medium and matched
for time in culture with the F442A cells. Figure 1
Saye et al Angiotensinogen mRNA in 3T3-F442A Cells
869
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FIGURE 1. Photomicrographs showing changes in F-actin fibers during spontaneous differentiation of
F442A cells. Left panels are phase-contrast photomicrographs of cells, whereas right panels show the F-actin
fibers from the same field visualized with rhodamine-phallocidin and a fluorescent filter. All photographs were
taken at x.400. Panels A and B: Preconfluent cells with the characteristic fibroblastlike morphology with
highly developed and well-organized F-actin fibers. Panels C and D: Confluent, contact-inhibited cells that
show no morphological changes associated with differentiation and have well organized F-actin fibers. Panels
E and F: Heterogenous cell population from a culture that had approximately 60% lipid-containing cells; most
of the cells have become spherical, and many have accumulated lipid droplets. Stress fibers lack the parallel
linear arrangement seen in panel D.
illustrates the transformation of the F-actin fibers in
the F442A cells from preconfluency to confluency to
spontaneously differentiated adipocytes. Preconfluent cells stained for F-actin showed a welldeveloped stress-fiber pattern with numerous parallel fibers. This pattern is also seen in the confluent
but undifferentiated cells (Figure 1, panels C and D).
As the F442A cells became confluent and contact
inhibited, cells began to retract their processes and
round up, an indication that differentiation had
begun.10 This was paralleled by a disruption of the
highly organized pattern of the F-actin fibers. As cells
continued to undergo phenotypic modulation and
began to accumulate lipid droplets, the F-actin fibers
were disassembled and became very disorganized.
This correlates with the decrease in actin in differentiated adipocytes.78 This is illustrated in Figure 1
(panels E and F) where many cells have become
rounded and have accumulated lipid droplets; concomitantly, the F-actin fibers (Figure 1, panels E and
F) appear to be undergoing disassembly, and many of
the remaining fibers no longer exhibit the parallel
linear arrangement found in undifferentiated cells.
When cells were allowed to differentiate spontaneously during long-term culture, morphological changes
were usually observed first. Random cells then began
to accumulate lipid droplets. As the percentage of
F442A cells containing lipid droplets increased, a flask
of F442A cells and a flask of control C2 cells were
removed for extraction of RNA. Angiotensinogen
mRNA accumulation increased as the percentage of
cells containing lipid droplets increased (Figure 2).
When approximately 20% of the cells contained lipid
droplets (as assessed by microscopic examination),
angiotensinogen mRNA was detected at low levels. In
Figure 2, lane 7 represents approximately 60% differentiation and correlated with the changes in F-actin
fibers illustrated in Figure 1 (panels E and F). As seen
in Figure 2, the expression of angiotensinogen mRNA
was increased dramatically with complete differentiation of F442A cells and did not change over the same
time course in C2 cells.
GPD is an established marker of differentiation in
many adipocyte model systems.5-7 To establish if the
angiotensinogen gene was regulated by the same
phenotypic modulatory events that regulate GPD, we
examined RNA extracted from two sets of spontaneously differentiated F442A cells (Figure 3). Densitometric analysis of dot blots probed for angiotensinogen and GPD revealed that the message levels for
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Vol 15, No 6, Part 2, June 1990
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both proteins increased approximately the same
amount in the spontaneously differentiating F442A
cells. When dot blots from time-matched C2 cells
were probed for angiotensinogen and GPD, there
were no detectable signals from either cDNA probe
(data not shown).
1.00
I 0.75
<D
U
cto
o 0.50
S
i
0.00
0
10 20 30 40 50 60 70 B0 90
Percent Differentiation
100
FIGURE 3. Plotting showing comparison of changes in
angiotensinogen (Ao) messenger RNA (mRNA) and gfycerol
phosphate dehydrogenase (GPD) mRNA during spontaneous
differentiation of 3T3-F442A cells. Autoradiographs of dot
blots were analyzed by an LKB Utroscan XL (LKBPharmacia, Piscataway, New Jersey), and values are expressed
as relative absorbance units. Circles represent angiotensinogen
mRNA, and squares represent GPD. Open symbols represent
densitometric analysis of dot blots from one set of spontaneously differentiating F442A cells, and closed symbols represent
data derived from a second experiment. Cells were from
different passages, and experiments were performed at different
times. The initial concentration of total RNA per sample was
1.0 fig, and each sample was serially diluted for each subsequent application to the blot.
FIGURE 2. Autoradiographic
detection of changes in angiotensinogen messenger RNA during
spontaneous differentiation of
3T3-F442A cells as compared
with time-matched C2 cells. Five
micrograms of total cellular RNA
in each lane was electrophoresed,
blotted, and hybridized to 100 ng
phosphorus-32-labeled 1.6 kb
angiotensinogen complementary
DNA (specific activity, 3.0x10?
dpm/iig). Exposure time was 3
days in the presence of an image
intensifying screen. Lanes 1, 3, 5,
7, 9, 11, and 13 represent RNA
from differentiating F442A cells
with approximately 20%, 30%,
50%, 60%, 75%, 85%, and 95%
lipid-containing cells within a
culture, respectively. Lanes 2, 4,
6, 8, 10, 12, and 14 represent
RNA from time-matched C2 cells
that do not differentiate. Lane 15
contained 5 jug o/ total RNA
from rat liver.
Discussion
Treatment of control 3T3-C2 cells and 3T3-L1 cells
with dexamethasone and IBMX to induce differentiation was shown to increase angiotensinogen mRNA
in adipocytes and undifferentiated C2 cells.4 Part of
the increase in angiotensinogen mRNA in adipocytes
was because of dexamethasone stimulation of the
angiotensinogen gene11 and was reflected by the
increase in mRNA seen in the C2 cells. The additional
increase in angiotensinogen mRNA in LI cells over
C2 cells, however, was postulated to reflect regulation
by the events that occur during the differentiation
process. To address this issue, we compared the
changes in angiotensinogen mRNA with those of
GPD mRNA and cytoskeletal elements as F442A cells
differentiated spontaneously. GPD represents one of
several cDNA clones isolated that increases as preadipocytes differentiate into adipocytes,5"7 whereas
F-actin mRNA decreases as the number of fat cells
increases within a culture.5"8 Changes in the F-actin
fibers and GPD mRNA correlated with changes in
angiotensinogen mRNA, indicating that the differentiation process might play a major role in the increased
accumulation of angiotensinogen mRNA.
Ringold et al12 have isolated cDNAs from TA1
adipocytes that represent mRNAs up-regulated by the
differentiation process. These mRNAs were divided
into two distinct classes as follows: Class I represents
RNAs expressed solely in the differentiated adipocyte,
whereas class II clones represent RNAs expressed at
low levels in preadipocytes that increase several times
on differentiation to adipocytes.12 Angiotensinogen
appears to belong to this second class of genes. Addi-
Saye et al Angiotensinogen mRNA in 3T3-F442A Cells
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tionally, Ringold et al12 have identified a class II clone,
clone 5, that, like angiotensinogen, can be regulated by
steroids in the preadipocyte and the adipocyte. Clone 5,
however, does not encode angiotensinogen because it
hybridizes to a 1.1 kilobase mRNA, whereas angiotensinogen mRNA is 1.8 kb. We have identified another
mRNA (angiotensinogen) in adipocytes that is
increased by the differentiation process and can be
regulated by steroids in both the undifferentiated and
differentiated state.
The role of angiotensinogen in preadipocytes and
adipocytes is unknown. Developmental regulation of
angiotensinogen gene expression suggests that angiotensinogen or the angiotensin peptides might have a
functional role in the differentiated cell. Although it
has not been demonstrated that renin is synthesized by
adipocytes, Schelling et al13 have reported reninlike
activity in lysates of wild-type 3T3 cells. We have
detected angiotensin I—like immunoreactrvity in the
media from 3T3-L1 and 3T3-F442A cells in the
absence of exogenous renin (unpublished observations
from our laboratory). Thus, it appears that adipocytes
are capable of processing angiotensinogen to angiotensin peptides. Because angiotensin II stimulates the
production of prostaglandins in isolated epididymal
adipocytes,14-15 these observations suggest that a paracrine angiotensin system might exist to regulate some
aspect or aspects of white adipose tissue function.
Lee et al16 have suggested a possible role for
angiotensinogen in the early development of the rat.
This is based on their findings that angiotensinogen
mRNA was detected at low levels in embryo bodies
and yolk sac on day 11 of embryogenesis and increased
until a plateau was reached at day 17. This angiotensinogen mRNA was about 200 nucleotides larger than
that expressed in adult liver. The authors hypothesized that most of the angiotensinogen mRNA
expressed in the body of the embryo was of hepatic
origin.16 A study by Gomez et al,17 however, identified
the perirenal and interscapular brown fat and kidney
as the sources of embryonic angiotensinogen mRNA.
Fetal liver angiotensinogen mRNA was detected at
day 20 of gestation only with poly A+-enriched liver
RNA. 17 Both studies detected low levels of angiotensinogen mRNA in fetal brain on day 15, which
increased significantly by day 20.16'17 Lee et al16 postulated that the increase in angiotensinogen mRNA in
fetal brain might be associated with the delayed
differentiation of nervous tissue. Thus, expression of
the angiotensinogen gene is tissue specific during
ontogeny and probably is regulated by the processes
associated with differentiation of at least some of the
tissues.
Therefore, in spontaneously differentiating 3T3F442A cells, F-actin fibers are disassembled as the
phenotypic expression of the cells is modified. Concomitantly, angiotensinogen mRNA levels increase as
the percentage of fat cells increases within a culture,
whereas no change occurs in the time-matched C2
cells. The changes in angiotensinogen mRNA levels in
spontaneously differentiated F442A cells are similar
871
to the changes in GPD mRNA, an established marker
of adipocyte differentiation. Thus, the increases in
angiotensinogen mRNA in spontaneously differentiating cells parallel both the structural changes associated with adipocyte differentiation and the increases
in GPD mRNA. We conclude that the expression of
angiotensinogen mRNA in spontaneously differentiating 3T3-F442A cells appears to be controlled by a
developmental regulatory mechanism.
References
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using hybridization in situ. Endocrinology 1987;121:1616—1626
2. Cassis LA, Lynch KR, Peach MJ: Localization of angiolensinogen messenger RNA in rat aorta. Girt Res 1988;62:1259-1262
3. Cassis LA, Saye JA, Peach MJ: Location and regulation of rat
angiotensinogen messenger RNA. Hypertension 1988;11:591-596
4. Saye JA, Cassis LA, Sturgill TW, Lynch KR, Peach MJ:
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KEY WORDS •
adipose tissue
angiotensinogen
actins
differentiation markers
Changes in angiotensinogen messenger RNA in differentiating 3T3-F442A adipocytes.
J Saye, K R Lynch and M J Peach
Hypertension. 1990;15:867-871
doi: 10.1161/01.HYP.15.6.867
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