Jf. Cell Set. i s , i-i5(i974)
Printed in Great Britain
LOCALIZATION OF CALCIUM IN THE SMOOTH
ENDOPLASMIC RETICULUM OF RAT
ISOLATED FAT CELLS
C. N. HALES, J. P. LUZIO
Department of Medical Biochemistry, The Welsh National School of Medicine,
Heath Park, Cardiff CF4 4XN, Wales
J. A. CHANDLER
Tenovus Institute for Cancer Research, Heath Park, Cardiff, CF\ \XX, Wales
AND L. HERMAN
Department of Pathology, State University of New York, Downstate Medical
Center, Brooklyn, New York 11203, U.S.A.
SUMMARY
The cytoplasm of the rat isolated fat cell contains a highly organized, interconnected system
of smooth endoplasmic reticulum having close association with the central lipid mass, mitochondria and cytoplasmic lipid droplets. Elements of smooth endoplasmic reticulum approach,
but do not fuse with, the plasma membrane.
When fat cells were treated with potassium pyroantimonate during fixation for electron
microscopy a precipitate was produced inside the smooth endoplasmic reticulum. Analysis with
an AEI-EMMA 4 analytical electron microscope showed that the precipitate contained calcium
but not sodium, magnesium or manganese.
It is possible that the smooth endoplasmic reticulum of fat cells may be structurally and
functionally analogous to the sarcoplasmic reticulum in skeletal muscle, and that redistribution
of calcium from a calcium store inside the smooth endoplasmic reticulum may be a consequence
of the action of lipolytic hormones.
INTRODUCTION
Rat isolated white fat cells have been shown to respond to a wide variety of
hormones (Rodbell, 1970). It is generally accepted that the effects of lipolytic hormones on such cells are mediated via a rise in intracellular cyclic AMP concentration,
and that anti-lipolytic hormones prevent such a rise (Robison, Butcher & Sutherland,
1971). As a result of experiments on the effects on lipolysis of local anaesthetics (Hales,
1970; Hales & Perry, 1970; Siddle & Hales, 1972), and glucocorticoids (Werner, Aim
& Low, 1972; Exton et al. 1972), and on EGTA depletion of fat cell calcium (Aim,
Efendic & Low, 1970; Efendic, Aim & Low, 1970), it has been suggested that hormonal control of lipolysis may be affected by alteration of the intracellular Ca2+
concentration in addition to alteration of cyclic AMP levels (Rasmussen, 1970; Siddle
& Hales, 1972). Insulin activation of rat fat cell pyruvate dehydrogenase may also
depend on an alteration of intracellular Ca2+ distribution (Martin, Denton, Pask &
Randle, 1972). In a number of other systems Ca2+ has been proposed as a hormone
'second messenger', additional to cyclic AMP (Rasmussen, 1970; Rasmussen, Goodman & Tenenhouse, 1972).
I
C E L 15
2
C. N. Hales, Jf. P. Luzio, J. A. Chandler and L. Herman
The cytosol concentration of Ca2+ in most mammalian cells appears to be low, with
Ca2+ apparently being stored elsewhere in the cell at a much higher concentration
(Rasmussen et al. 1972). Hormones may alter cytosol Ca2+ concentration, either by
affecting the Ca2+ permeability of the plasma membrane or by regulating its uptake
and release from an intracellular store (Rasmussen, 1970; Rasmussen et al. 1972).
The current study was undertaken to investigate the possibility of hormonally
induced changes in the ultrastructure of rat isolated fat cells and to look for sites of
intracellular ion localization. Since it was initiated, several publications describing the
results of ultrastructural studies of rat isolated fat cells have appeared (Pictet et al.
1968; Schotz et al. 1969; Cushman, 1970; Slavin, 1972). It is the purpose of this
paper to extend these findings and to report an investigation of the cation content of
the smooth endoplasmic reticulum. We present the hypothesis that part of the smooth
endoplasmic reticulum of fat cells may be structurally and functionally analogous to
the sarcoplasmic reticulum in skeletal muscle.
MATERIALS AND METHODS
Male Wistar rats (120-140 g), fed on a stock laboratory diet, were used without previous
starvation. Rats were killed by decapitation, and fat cells prepared from the epididymal fat
pads by the method of Rodbell (1964). Isolated cells were washed and suspended in KrebsRinger bicarbonate buffer (1-3 x io~ 3 M Ca 2+ ), pH 7-4 (Cohen, 1957), containing 4 % bovine
serum albumin. On some occasions, prior to fixation, cells were incubated in this buffer for 1 h
at 37 °C in the presence of various test substances. All glassware with which fat cells came into
contact was treated with Siliclad (Clay Adams, Parsippany, N.J., U.S.A.) before use.
The fat cell suspension was centrifuged (joog, 30 s) and the infranatant removed. For
morphological investigation cells were fixed for 30 min at room temperature in 0 1 M cacodylate
buffer, pH 7 4 containing 2 5 % glutaraldehyde (Sabatini, Bensch & Barrnett, 1963), (Polysciences Inc., Rydall, Pennsylvania), and washed 3 times with cacodylate buffer containing
0 2 M sucrose. The fixed cells were post-osmicated for 30 min in 0-14 M veronal-acetate buffer,
p H 7 4 containing 1% OsO 4 (Palade, 1952), followed by suspension in veronal-acetate buffer
containing 5 % uranyl acetate. The cells were then dehydrated in ascending concentrations of
ethanol, washed repeatedly in propylene oxide until no further black material was leached out
and embedded in Epon. Thin sections were cut on an LKB Ultrotome III, mounted on
uncoated copper grids, stained with a saturated solution of uranyl acetate in 50 % ethanol
(prepared in the dark), washed with water, stained with tartrate-stabilized lead hydroxide
(Millonig, 1961), and again washed with water. Stained sections were examined and photographed with an AEI EM801 electron microscope, using the tilt stage where necessary.
For antimonate localization of cations in fat cells, the method of fixation and staining was
modified from that described above. The method of antimonate precipitation used was developed
by 2 of the current authors in a study of cation localization in pancreatic islets of Langerhans
(Herman, Sato & Hales, 1973), by modification of the technique of Spicer et al. (1968). Although
the pyroantimonate precipitation technique was originally devised for sodium localization,
sodium is rapidly lost from fat cells in normal medium (Perry & Hales, 1969), the presence of
phosphate ions may interfere with the formation of sodium pyroantimonate complexes (Torack
& Lavalle, 1970), and pyroantimonate has been shown to precipitate other cations, this lack of
specificity making subsequent elemental analysis essential (Herman et al. 1973). Cells were
fixed for 30 min at room temperature in 25 % glutaraldehyde buffered to pH 7 4 with 0 1 M
potassium phosphate, and washed 3 times with potassium phosphate buffer containing 10%
sucrose. The fixed cells were left overnight in the final buffer wash and then post-osmicated for
30 min in 1 % osmium tetroxide-2 % potassium pyroantimonate (BDH). The cells were subsequently washed 3 times in 0 1 4 M veronal—acetate buffer, pH 7 4 and incubated for 2 h in
veronal-acetate buffer containing 5 % uranyl acetate. The fixed, stained cells were dehydrated,
embedded and sectioned as described above. Thin sections were mounted on carbon-coated
Localization of calcium in rat fat cells
3
copper grids, examined and photographed in an AEI EM801 electron microscope, and also
examined in an AEI-EMMA 4 analytical electron microscope to enable elemental analysis of
antimonate deposits. Details of the theory and operation of the analytical electron microscope
have been discussed by Marshall & Hall (1966), Cooke & Duncumb (1969), Chandler (1971,
1973), and Weavers (1973).
Examinations of the precipitates were made with EMMA for calcium, manganese, sodium
and magnesium. Positive readings were found only for calcium and the conditions of operation
chosen were: 100 kV, probe diameter o-i fim, probe current 0 0 1 - 0 0 3 /*A. Counting time was
100 s at each point of analysis. The crystal spectrometers were used to detect calcium since the
energy dispersive analyser was unable to resolve the peaks of calcium and antimony (CaK« =
3691 keV, SbKe< = 3604 keV). The pentaerythritol (PET) crystals were chosen with one
spectrometer being set permanently on peak position and the other on background. A given
number of precipitates at a given number of different cell foci were measured and the mean
values presented to demonstrate relative calcium distribution (see Table 1 with reference to
Fig. 1, p. 4). The precipitates varied in size at each place but for quantitative comparison the
same probe area was used at each place. The shape of the probe was varied from circular to
elliptical without changing the probe current, simply by adjustment of the microscope stigmator.
In addition, analyses were made on sections that had not been fixed with the antimony salt
solution.
RESULTS
Incubation of the isolated fat cells with various hormones (adrenalin, io/tg/ml;
ACTH, 10/tg/ml; insulin, 50/iU/ml), and other substances (dibutyryl cyclic AMP,
5 mivi; theophylline, 1 mM; propranolol, 10/tg/ml), prior to fixation produced no
apparent overall changes in the morphology of the cells. Photographs of fat cell
sections shown in the current paper were chosen for clarity of the image, irrespective
of the pre-fixation treatment of the cells.
The overall morphology of the rat isolated white fat cell was observed to be in
accordance with previously published work (Rodbell, 1967; Schotz et al. 1969;
Cushman, 1970; Slavin, 1972). The extensive membrane system with the appearance
of smooth-surfaced endoplasmic reticulum (ER) and its interesting relationships to
other subcellular organelles prompted further electron-microscopic investigation.
Smooth endoplasmic reticulum and the central lipid droplet
The central lipid droplet was observed to be surrounded by a smooth membrane
system previously described as saccules of smooth endoplasmic reticulum (Pictet et al.
1968), as a fenestrated double membrane (Schotz et al. 1969), or as a fenestrated
envelope (Cushman, 1970). This smooth membrane system appeared to be part of a
highly organized, interconnected system of smooth ER found throughout the cytoplasm and having close association with mitochondria and cytoplasmic lipid droplets
as well as with the central lipid mass (Figs. 2, 4). The smooth ER surrounding the
central lipid mass could often be followed round the entire portion of the cell present
in the section being examined, but was apparently not immediately applied to the lipid.
The region of the interface between the cytoplasm and the central lipid mass is
clearly one of great functional significance since it is probably the site at which lipid
deposition and mobilization take place. Careful ultrastructural study of this region
revealed 2 different structures, an electron-opaque line (Figs. 2-4), or thin filaments
entering the lipid droplet as previously described by Schotz et al. (1969). The relationship between these alternative structures was not resolved.
4
C. N. Hales, J. P. Luzio, J. A. Chandler and L. Herman
Smooth endoplasmic reticulum and mitochondria
A feature of the smooth ER system was its close association with mitochondria.
Smooth ER was frequently observed not only to pass close to mitochondria, but also
to encircle them (Figs. 2, 4).
Smooth endoplasmic reticulum and the plasma membrane
In many sections smooth ER was seen to approach the plasma membrane. A close
relationship of the 2 membranes was observed, both at sites of invagination of the
plasma membrane (Fig. 4), and when no such invagination occurred (Figs. 2, 3).
Whenever smooth ER appeared to fuse with the plasma membrane the section was
further examined using the tilt stage. With this technique it was found that, in every
case examined, the smooth ER and plasma membranes were discrete and did not fuse
(Fig- 3)Central
lipid mass
Dense
Fine
precipitation
Fig. 1. Schematic representation of regions (A-I) of fat cell analysed with EMMA.
Intracellular cation localization
After fixation of fat cells in glutaraldehyde and postfixation in osmium tetroxide/
pyroantimonate a dense precipitate was observed in thin sections (Figs. 5-7). This
precipitate appeared to be mainly localized adjacent to the central lipid droplet
(Fig. 5). Mitochondria and nuclei were usually free of precipitate. In a few sections
precipitate was associated with the plasma membrane (Fig. 6). Examination of the
precipitate at higher magnification revealed that some of the precipitation particles
were deposited in the smooth ER system (Fig. 7). In some sections lines of dense
precipitate ran into the cytoplasm away from the central lipid droplet (Fig. 6), the
pattern being very similar to that observed for smooth ER (Fig. 2). Fine precipitate,
randomly scattered and not associated with any organelle, was observed in the
Localization of calcium in rat fat cells
5
ground substance of the cytoplasm (Figs. 6, 7). The significance of this precipitation
is uncertain since it varied considerably from one preparation to another. No correlation of this deposition was noted with any ultrastructural feature.
Fig. 1 represents schematically the regions of the fat cell analysed with EMMA.
Calcium was the only cation found to give positive readings using EMMA; magnesium, manganese and sodium were undetectable. A limitation of the analysis was that
precipitates varied in size and were always smaller than the probe diameter (o-i /(.m).
Nevertheless the results indicate a specific distribution of calcium localization as
shown in Table 1. The values represent calcium X-ray counts averaged over a number
of areas of the same type in a counting period of 100 s. No attempt was made to assess
mass fractions of calcium in the tissue sections because of the presence of heavy
metal stains. Within each section X-ray counts are directly proportional to local
calcium mass values, and assuming constant section mass thickness, represent relative
calcium concentration values.
Table 1. Calcium X-ray counts for pyroantimonate-treated rat fat cells
Area of fat cell analysed with EMMA
A. Dense precipitate at the interface of cytoplasm
with central lipid droplet
B. Dense precipitate within smooth ER
adjacent to the central lipid droplet
C. Precipitate associated with central lipid droplet
D. Cytoplasmic matrix
E. Plasma membrane
F. Mitochondria
G. Interface of cytoplasm and central lipid
droplet lacking precipitate
H. Central lipid droplet lacking precipitate
I. Precipitate external to plasma membrane
X-ray counts in 100 s,
mean + S.E.M.
n
P
286 ± 4 9
18
001
24-6 ±
2I-O ±
12-3 +
8-o +
i6-s ±
3-9
3-6
3-8
1'9
7-6
7
< o-ooi
9
OOI
7
8
005
005
4
OIO
13-7 + 3-2
i-8 + 2-2
7
OOI
± 9'7
5
326
6
OOI
X-ray counts normalized to 001 /iA beam current.
n, number of observations at each cell area.
P, significance of the difference from the X-ray counts in area H estimated by Student's t test.
DISCUSSION
Cushman (1970) commented on the extensive smooth-surfaced endoplasmic reticulum of the rat isolated fat cell. The current study shows the ultrastructural interrelationship of this system with the central lipid droplet, mitochondria and the plasma
membrane. It is difficult to believe that the striking morphology of this highly developed system does not have important implications for the function of the tissue.
A similarly highly organized system of smooth membranes is observed in skeletal
muscle (the sarcoplasmic reticulum), and in terms of regulation adipose tissue may be
considered to pose similar anatomical problems to skeletal muscle. In both tissues it is
believed that the effect of substances acting at the plasma membrane is transmitted to
a centrally situated mass with a clearly defined and regular geometry in relation to the
6
C. N. Hales, J. P. Luzio, J. A. Chandler and L. Herman
plasma membrane. In skeletal muscle, this mass is the contractile protein activated by
events at the neuromuscular junction. Activation of contraction has been shown to be
mediated via release of Ca2+ from the sarcoplasmic reticulum (Ebashi, Endo &
Ohtsuki, 1969). Localization of calcium has been demonstrated in the sarcoplasmic
reticulum of skeletal muscle using oxalate precipitation (Constantin, Frazini-Armstrong & Podolsky, 1965), and also by the technique of pyroantimonate precipitation
in conjunction with X-ray micro-analysis (Yarom & Chandler, 1972).
In adipose tissue, hydrolysis of triglyceride is activated by a number of hormones,
most of which are currently believed to have their primary action at the plasma
membrane (Rodbell, 1972). Subsequent to binding to the plasma membrane, the
hormones stimulate intracellular production of cyclic AMP. This in turn may activate
lipolysis by mechanisms analogous to those mediating the activation of glycogenolysis
(Hales, Chalmers, Perry & Wade, 1968; Wade, Chalmers & Hales, 1970; Huttunen,
Steinberg & Mayer, 1970; Huttunen & Steinberg, 1971). However, results indicating
the importance of ions in the regulation of lipolysis have also suggested that a rise in
intracellular cyclic AMP concentration is not in itself enough to activate lipolysis
(Hales et al. 1968; Hales & Perry, 1970; Siddle & Hales, 1972). As discussed in the
Introduction, there is considerable evidence to suggest that alteration of cytosol
calcium concentration may be required for control of lipolysis, and the possibility that
an intracellular store of calcium may be mobilized during hormonal stimulation of
lipolysis has been suggested (Rasmussen, 1970; Efendic et al. 1970). In experiments
with rat isolated fat cells it has not been possible to show that extracellular calcium is
essential for catecholamine-stimulation of lipolysis. Removal of calcium from the
extra-cellular medium has been shown to diminish adrenalin-stimulated lipolysis
only at low hormone concentrations (Mosinger & Vaughan, 1967). More recently
Efendic et al. (1970) found that preincubation of human adipose tissue pieces in
calcium-free medium in the presence of the calcium chelator EGTA increased the
inhibition of noradrenalin-stimulated lipolysis produced simply by addition of EGTA
to the incubation medium. Moreover, although theophylline- and dibutyryl cyclic
AMP-stimulated lipolysis were not inhibited by the presence of EGTA in the
incubation medium, preincubation with EGTA resulted in inhibition. These authors
suggested that preincubation of adipose tissue pieces with EGTA depleted endogenous
stores of calcium which could be mobilized in the stimulation of lipolysis. In searching
for a structural basis for such an intracellular calcium pool we have used the pyroantimonate precipitation technique previously employed to localize calcium in the
pancreatic /? cell (Herman et al. 1973), and we have shown calcium localization in the
smooth ER of the rat isolated fat cell.
It may appear surprising that using the pyroantimonate X-ray microanalysis technique, calcium was not found to be associated with other intracellular structures, for
instance mitochondria, which in most tissues have the ability to absorb and store
calcium. In addition, calcium localization within the smooth ER system was localized
mainly to that ER close to the central lipid droplet. Several explanations might be
offered for these apparent anomalies. The technique is likely to be dependent both on
whether calcium is complexed or free within cellular structures, and on the methods
Localization of calcium in rat fat cells
7
used for fixation, staining and dehydration. Similarly, it is possible that other
cations present in the cell do not precipitate with pyroantimonate. It has been suggested that the presence of phosphate ions (as used in the current investigation), might
interfere with the formation of sodium pyroantimonate complexes (Torack & Lavalle,
1970; Tisher, Weavers & Cirksena, 1972), and also that electron microscopy fixatives
may affect cellular accumulation of cations (Krames & Page, 1968; Yarom, Ben-Ishay
& Zinder, 1972). Perry & Hales (1969) have reported that the half time of efflux of
intracellular 22Na+ from rat isolated fat cells into normal medium was 28 min.
Despite these problems it seems clear that at least part of the smooth ER system of the
rat fat cell contains calcium as a predominant cation.
Microsomal fractions prepared from muscle, liver, kidney and brain have been
shown to be capable of ATP-dependent calcium uptake (Yoshida, Kadota & Fujisawa,
1966; Ohtsuki, 1969; Diamond & Goldberg, 1971). If fat cell smooth ER is similarly
capable of calcium transport and storage, it is possible that, as in muscle (Ebashi et al.
1969), the close proximity of elements of the smooth ER system to the plasma membrane may allow activation of smooth ER calcium uptake or release as a response to
events at the cell surface. Such an activation may be effected by cyclic AMP. In
muscle theophylline can stimulate Ca2+ release by the sarcoplasmic reticulum (Weber
& Herz, 1968).
Thus, there may be a structural basis, not only for a mobilizable calcium store within
the fat cell, but also for a mechanism of stimulating mobilization as a result of events
at the cell surface. The existence of such a mechanism would be of obvious importance
if alteration of cytosol Ca2+ concentration is indeed necessary for the mediation of
hormone action on fat cells. The observed close association of smooth ER with mitochondria agrees with earlier observations of a close topographical relationship between
mitochondrial membranes and other cytoplasmic membranes both in adipose tissue
and other tissues (Sheldon et al. 1962; Bernhard & Rouiller, 1956; Sheldon, 1956).
This relationship may also be important in control of cytosol Ca2+ concentration,
since in muscle there is considerable evidence that mitochondria play an important
role in its control (Rasmussen et al. 1972; Carafoli & Azzi, 1972).
The initial part of this work was carried out in the Department of Biochemistry, University
of Cambridge. We are grateful to Professor Sir Frank Young for the facilities and encouragement he provided at that time. Professor D. H. Northcote and Dr F. B. P. Wooding provided
much helpful guidance and discussion. We are also grateful to Margaret Hodgkins, Diana
Jones, Gillian Rowe and Gwyneth Parry for their skilled technical assistance and to Len Jewitt
and Ralph Marshall for the photography. The work was supported by grants from the British
Diabetic Association, Science Research Council, the Wellcome Research Foundation, and by the
Tenovus organization. J.P.L. held a studentship for training in research methods from the
Medical Research Council and L.H. was supported by USPHS Research Grant CA-06081,
USPHS Special Fellowship AM 39072 and SUNY Research Foundation Grant-in-Aid 71062.
Part of this work is to be submitted for a Ph.D. degree in the University of Wales by J.A.C.
8
C. N. Hales, J. P. Luzio, J. A. Chandler and L. Herman
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{Received 30 August 1973)
Fig. 2. Electron micrograph of a portion of isolated rat fat cell cytoplasm showing the
smooth endoplasmic reticulum system {er). I, central lipid droplet; m, mitochondrion;
pm, plasma membrane. The elements of the smooth endoplasmic reticulum system
close to the central lipid droplet are apparently continuous (arrows) with those
elements running throughout the cytoplasm, close to the mitochondria and approaching the plasma membrane (double arrow), x 45000.
Fig. 3A-C. Portion of isolated rat fat cell cytoplasm showing an element of smooth
endoplasmic reticulum (er) closely approaching the plasma membrane (pm). I, central
lipid droplet. The 3 micrographs represent different tilt stage angles: A, untilted;
B, 20° right; and C, 20° left. The use of the tilt stage shows that this element of smooth
endoplasmic reticulum does not fuse with the plasma membrane, x 90000.
Localization of calcium in rat fat cells
3A
3B
II
12
C. N. Hales, J. P. Luzio, J. A. Chandler and L. Herman
Fig. 4. Elements of smooth endoplasmic reticulum {er) surround a mitochondrion
(m) in a portion of isolated rat fat cell cytoplasm. /, central lipid droplet; pm, plasma
membrane, x 100000.
Localization of calcium in rat fat cells
14
C. N. Hales, J. P. Luzio, J. A. Chandler and L. Herman
Fig. 5. Electron micrographs of isolated rat fat cells after glutaraldehyde-osmiumpyroantimonate fixation. /, central lipid droplet; m, mitochondrion; pm, plasma
membrane. There is a linear array of irregularly placed, dense precipitate adjacent to
the central lipid droplet of each cell, x 30000.
Fig. 6. Portion of isolated rat fat cell cytoplasm after glutaraldehyde-osmiumpyroantimonate fixation. /, central lipid droplet; pm, plasma membrane. There is
dense precipitate along the lipid droplet which appears to extend into the cytoplasm
(arrows), as well as precipitate in association with the plasma membrane, x 120000.
Fig. 7. Portion of isolated rat fat cell cytoplasm after glutaraldehyde—osmium—pyroantimonate fixation. /, central lipid droplet; pm, plasma membrane. The dense precipitate is bound by membrane (arrows) of smooth endoplasmic reticulum. x 120000.
Localization of calcium in rat fat cells