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Fine structural studies on white adipocyte differentiation.

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Fine Structural Studies on White Adipocyte Differentiation
Department of Anatomy, University o f Southern California School of Medicine,
Los Angeles County- USC Medical Center, Los Angeles, California 90033
The differentiation of mammalian white adipocytes from prenatal through early postnatal periods was studied by light and electron microscopy in C57BL mice. Anatomical regions chosen for this study were the epididymal, mesometrial, mesenteric and inguinal fat pads. In each of these
regions, adipocytes differentiated from fibroblast-like cells (preadipocytes)
characterized by a n ovoid nucleus, profiles of rough endoplasmic reticulum, microtubules, microfilaments, spherical mitochondria, and small multiple lipid inclusions. Preadipocytes of the inguinal fat pad were first observed prior to birth
(17-19 days), whereas, in the other anatomical sites, these cells were not observed until one to three days postnatally. As differentiation proceeded, and as
the adipocytes assumed a spherical shape, there was a progressive decrease in
the amount of rough endoplasmic reticulum and microfilaments concomitant
with transient glycogen storage and a n increase in the size of lipid droplets.
Mature unilocular adipocytes were observed in the inguinal fat pads a t three
days of age. On the other hand, these cells did not appear until seven days after
birth in the epididymal fat pad, mesometrium and mesentery. Regardless of the
anatomical region studied, the differentiation of preadipocytes to adipocytes
proceeded similarly. Preadipocytes could not be distinguished from fibroblasts
morphologically within the fat depots studied. Adipocytes a t the mid-stages of
differentiation and in all regions studied occasionally exhibited close intercellular contacts of varying morphology.
The differentiation of mammalian white
adipocytes has been extensively studied by
light microscopy with considerable debate
concerning the nature of the adipocyte precursor (Clark and Clark, '40; McCullough, '44;
Tedeschi, '60; Barrnett, '62; Wasserman, '64;
Simon, '65; Tavassoli, '76). Napolitano ('63)
was the first investigator to demonstrate by
electron microscopy t h e development of
adipocytes in the rat. Recently, Desnoyers and
Vodovar ('77) compared the fine structural development of adipocytes in pigs and rats. In
these studies, the adipose depots were fixed
solely in osmium tetroxide. The present paper
reevaluates t h e specific morphologic and cytologic changes occurring during adipocyte development by the use of primary aldehyde
fixation which allows for the demonstration
of several cytologic features previously undescribed.
Adipocytes from the epididymal, mesometrial, mesenteric and inguinal fat pads were
(1979)195: 63-72.
studied following routine immersion fixation
in 3% glutaraldehyde and 1%formaldehyde
buffered with sodium cacodylate (0.1 M), pH
7.2. The tissues were obtained from eight male
and eight female C57BL mice varying in age
from 17 days prenatal to 7 days postnatal.
Blocks of tissue were postfixed in 1%osmium
tetroxide, dehydrated in ethanol and embedded in Spurr's low-viscosity epoxy resin or in
an Epon-Araldite mixture. Routine thick sections (1 pm) were cut and stained with silver
according to the procedure of Rosenquist et al.
('71). Thin sections were cut on a LKB I11 ultramicrotome, stained with uranyl acetate
(Watson, '58) and lead citrate (Reynolds, '63)
and viewed in a Hitachi HUl2A electron microscope.
The maturation of adipocytes followed a
similar course in all fat pads examined. Light
Received July 8. '77. Accepted Dec. 29, '78.
I This work was supported by NIH Grants HL 18283 and GRS 5
SO1 RR05356.
microscopic examination revealed the adipocyte precursors to be spindle-shaped, and
containing relatively large, smooth, ovoid or
elongated nuclei (fig. 1). Later, the cells
enlarged and assumed an oval or rounded
shape as intracellular lipid droplets accumulated (fig. 2). Further on in development,
many cells contained single lipid droplets and
eccentrically placed nuclei (fig. 3).
Adipocyte development in the subcutaneous
inguinal fat pads commenced approximately
17 days prenatally. At birth, there was a mixture of multilocular and unilocular cells with
the greatest proportion being the multilocular. Unilocular adipocytes were in abundance
in this site two to three days after birth.
In contrast to the subcutaneous inguinal fat
pads, adipocyte development in the epididymal, mesometrial and mesenteric fat pads did
not commence until birth. Three days after
birth most adipocytes were of the multilocular
variety, whereas, by the seventh postnatal
day, most were unilocular. During the latter
two stages there was great variation in size
and shape of the developing adipocytes. Thus,
some adipocytes appear to require less than
seven days to reach the unilocular stage.
At the fine structural level, the adipocyte
precursor (preadipocyte) (fig. 4)was indistinguishable from fibroblasts noted in the thin,
non-fatty membraneous part of the mesentery. This cell was characterized by its spindle-like shape with several long tenuous processes protruding from both poles of the cell.
The cytoplasm contained free ribosomes and
rough endoplasmic reticulum whose cisternae
contained granular deposits. The few scattered mitochondria were mostly spherical and
possessed a simple internal structure. Flattened Golgi zones, a variety of smooth and
coated vesicles, microfilaments, and microtubules were also present. An external lamina
was not developed, although these cells were
noted to be in intimate contact with a n extracellular fibrillar matrix.
During the mid-stage of development (figs.
5, 6) adipocytes became spherical. Nuclei
rounded up, and there was a n increase in t h e
amount of intracellular lipid droplets. Glycogen granules began to appear throughout
t h e cytoplasmic matrix. The number of
micropinocytotic vesicles and mitochondria
had increased. Concomitantly, Golgi zones and
rough endoplasmic reticulum were quite flat
and devoid of granular material. An external
lamina had developed a t many sites.
Other features of developing adipocytes included peculiar intercellular contacts characterized by penetrating and interdigitating cellular processes (figs. 7, 8). The space between
such cellular contacts measured approximately 150 A. In these regions of surface contact,
the plasma membranes did not appear denser,
nor did they exhibit any specialization typical
of epithelial cells. Rarely, the plasma membranes of contiguous adipocytes assumed the
form of gap-like junctions.
Fully mature, unilocular adipocytes observed in this report were characterized by
their large size, eccentrically-placed nucleus,
large central lipid droplet, smooth-surfaced
vesicles and endoplasmic reticulum, free ribosomes, and numerous micropinocytotic vesicles. Profiles of rough endoplasmic reticulum
or glycogen particles were rare.
The description of the stages of adipocyte
maturation in the mouse reported in this
paper agrees with that reported for the rat by
Napolitano ('63). The adipocyte is derived
from a spindle-shaped cell resembling a fibroblast. It begins to deposit lipid in the form of
small droplets located a t one pole of the cell.
The adipocyte rounds up, deposits more lipid,
and synthesizes glycogen transientally. By
means of additional lipid deposition, the cell
enlarges and assumes the typical mature
signet-ring shape.
The preadipocytes observed in this study
differ from those described by Napolitano
('63) in that they contain microtubules, microfilaments, coated vesicles, free ribosomes and
prominent Golgi zones. The absence of a description of these organelles by the latter
author was probably due to the use of osmium
tetroxide fixation which has been shown to
cause protein extraction (Hayat, '70).
Although there has been much debate concerning the precise origin of the adipocyte
(Barrnett, '62; Napolitano, '63; Wasserman,
'641, the question still remains whether the
adipocyte is derived from the fibroblast or
from its own precursor. I found no morphologic
difference between the earliest preadipocytes
and typical fibroblasts in all the developing
depots studied (including the non-fatty part of
the mesentery). One could conclude that
adipocytes simply originate from fibroblast
precursors. However, Poznanski et al. ('731,
Van e t al. ('76), Dardick et al. ('761, and Van
and Roncari ('77) demonstrated in vitro that
human and rat preadipocytes from the subcutis and epididymis formed mature adipocytes (morphologically and biochemically)
while dermal fibroblasts cultured similarly
did not have the same capacity. Van and Roncari (’77) suggested t h a t adipocytes may develop from “specialized” fibroblasts. Although
the latter authors provided no explanation, it
could be that such cells undertake the role of
fibroblasts (i.e., synthesis of collagen) and
then are “programmed” to synthesize lipid
and become adipocytes. Thus, these specialized fibroblasts differ from typical fibroblasts
seen in tendons, ligaments, etc., where no fat
cells are produced.
Green and Meuth (’74) have established a
clonal line of preadipocytes (3T3-Ll) which
were derived from mouse 3T3 fibroblasts.
However, the possibility that the 3T3-Ll cells
are derived from vascular endothelium has
been suggested by Gospodarowicz et al. (’76).
In addition, Desnoyers and Vodovar (’77) presented morphologic evidence that adipocytes
in the pig and rat are derived from endothelial
cells. And Tavassoli (’76) showed that the precursors of bone marrow adipocytes are cells associated with the sinus endothelium.
Several instances of cell-to-cell contact including gap junctions between developing
adipocytes have been noted in this report. This
finding is in accord with the work of Sheridan
(’71)who demonstrated electrical coupling between amphibian white adipocytes and mammalian brown adipocytes. Obviously, a more
thorough study of white fat using freeze fracture techniques and electron-opaque tracers is
necessary to determine the frequency and
importance of such contacts.
The author wishes to thank Mr. Stephen
Harris for his outstanding technical assistance.
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The following three micrographs represent sections of mouse epididymal adipose tissue stained with silver nitrate. X 590.
1 Newborn. Single arrows point to spindle-shaped adipocyte precursors. Double arrows
indicate a cell t h a t appears to be rounding u p and depositing intracellular lipid.
2 Three days postnatal. Developing adipocytes have become ovoid or spherical. Nuclei
are eccentric. Lipod droplets (L) have increased in size.
Seven days postnatal. Most cells have enlarged due to an increase in the size of lipid
droplets (L). Nuclei are pushed to the periphery by the expanding lipid.
Bernard G. Slavin
Preadipocyte in t h e developing epididymal fat pad. Note abundance of rough endoplasmic reticulum (RER)containing amorphous granular material, microtubules
(MT), microfilaments (MF), lipid droplets (L), small spherical mitochondria (m), and
a Golgi zone (GI.The external lamina is not yet formed. Cell is surrounded by a
fibrillar matrix (FM). x 28,000.
Bernard G. Slavin
5 Inguinal adipose tissue, newborn mouse. Numerous lipid droplets (L) fill the cell.
Transient glycogen deposition (GLY) was observed. Note the large increase in number of mitochondria. X 5,600.
6 Higher power view of the multilocular stage of adipocyte development similar to
that seen in figure 5. Note t h e vast reduction in profiles of rough endoplasmic reticulum (ER). The external lamina (EL) is well developed a t this stage. Several micro-pinocytotic vesicles (MPV) are noted. Lipid droplets (L); mitochondria (m).
x 19,000.
Bernard G . Slavin
Bernard G. Slavin
7 , 8 Adipocytes of the inguinal fat pad of newborn mice. Note t h e penetrating and interdigitating processes (*) between adjacent cells. The intercellular space a t the
areas of close contact is approximately 150 A. Lipid droplet (L). x 39,000.
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adipocyte, structure, differentiation, white, studies, fine
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