From the
Steroidogenic cells store cholesteryl esters, precursors for
steroid hormone synthesis, in intracellular lipid droplets. Cholesteryl
ester hydrolysis is activated by protein kinase A and catalyzed by
cholesteryl esterase. The esterase is similar, if not identical, to
hormone-sensitive lipase in adipocytes where an analogous lipolytic
mechanism occurs. Perilipins, proteins located exclusively at lipid
droplet surfaces in adipocytes, are polyphosphorylated by protein
kinase A in response to lipolytic stimuli, suggesting a role for these
proteins in mediating lipid metabolism. The present study reveals that
perilipins are associated with cholesteryl ester droplets in two
steroidogenic cell lines: Y-1 adrenal cortical cells and MA-10 Leydig
cells. The relative abundance of perilipin mRNAs and protein is much
less in steroidogenic cells than in adipocytes. Like adipocytes,
steroidogenic cells express perilipin A; additionally, the latter cells
contain relatively abundant amounts of perilipin C, a protein that is
not detectable in adipocytes by Western analysis. The data suggest a
strong link between perilipins and lipid hydrolysis that is mediated by
the hormone-sensitive lipase/cholesteryl esterase class of enzymes.
Nature has devised a strategy for handling neutral lipids, such
as triacylglycerols and cholesteryl esters, in the extracellular
aqueous environment by packaging them into lipoproteins for delivery to
tissues via the blood. These well characterized conveyances have an
outer monolayer of polar lipids and proteins that serve specific roles
in nucleation of lipoprotein formation, targeting of lipoproteins to
specific cell surface receptors, and metabolism of the component lipids
(1, 2). While much is known about the structure and metabolism of
lipoproteins, little is known about the composition of two
metabolically important intracellular lipid pools: the
triacylglycerol-rich droplets in adipocytes that contain the vast
majority of bodily energy reserves and the cholesteryl ester-containing
droplets in steroidogenic cells that contain the precursors for steroid
hormone synthesis. Recently, we identified unique proteins, termed
perilipins, located exclusively at the surface of intracellular lipid
droplets of adipocytes(3, 4) . Based on their
subcellular location and metabolic properties, i.e. polyphosphorylation in concert with lipolytic stimulation of
adipocytes, we have suggested that perilipins play a role, as yet
undefined, in lipid metabolism. Although initial Western and Northern
blot analyses failed to reveal perilipins in non-adipose
tissues(3, 4) , striking parallels between the modes of
lipid hydrolysis in adipocytes and steroid producing cells led us to
reinvestigate the question of perilipin distribution. cAMP stimulates
lipid hydrolysis by hormone-sensitive lipase in adipocytes and by
cholesteryl esterase in steroidogenic cells; these hydrolytic enzymes
are similar, if not identical(5, 6, 7) . Given
these similarities in lipid metabolism, one might expect to find
perilipins associated with the lipid droplets in steroidogenic cells.
In the present paper, we report the occurrence of lipid
droplet-associated perilipins in both adrenal cortical and Leydig
cells, including one major species, perilipin C, that is relatively
abundant in these steroid producing cells but is not detectable by
Western blotting of adipocyte proteins.
The major form of perilipin expressed in primary rat and
murine adipocytes, and in cultured murine 3T3-L1 adipocytes, is
perilipin A, predicted to be
The two species of
perilipin, A and B, for which sequence data are available arise by
alternative splicing of RNA; A and B are identical through their 406
NH
Previously, with a probe containing full-length
coding perilipin A cDNA, we detected two perilipin mRNA species in
primary rat adipocytes by Northern blot analysis, one of 3.9 kb
corresponding to perilipin B and the other of 3.0 kb, which is
perilipin A(4) . With the same probe, four mRNA species were
detected in the four murine cell lines examined: primary and cultured
3T3-L1 adipocytes, Y-1 adrenal cells, and MA-10 Leydig cells. The
relative abundances of the four mRNAs differed among the cell lines (Fig. 2). The 3.0-kb mRNA was the most abundant in all cells, and
further studies with selective probes indicated that as in rat, this
species encodes perilipin A.
As in adipocytes, the major species
expressed in the steroidogenic cells is perilipin A, identified by
co-migration in SDS-PAGE gels with adipocyte perilipin A, by
recognition of the protein by antibodies directed against the unique
COOH-terminal segment of perilipin A, and by altered migration on
SDS-PAGE upon phosphorylation by PKA, corresponding to the migration of
adipocyte perilipin A in its PKA-phosphorylated form. The occurrence of
relatively abundant amounts of perilipin C in steroidogenic cells is in
contrast to adipocytes, where expression of perilipin C mRNA is minimal
and no perilipin C protein is detected. One factor that makes this
finding especially intriguing is the absence in perilipin C of the
COOH-terminal segment of perilipin A, which contains three of the six
consensus PKA sites (4). Thus, if phosphorylation of perilipin by PKA
is required for or facilitates lipid hydrolysis, perilipin C may
protect against lipid metabolism and thus provide a mechanism for fine
tuning of steroid hormone production. The question of similarity or
identity between cholesteryl esterase and hormone-sensitive lipase is
also unresolved. In accord with the findings of Holm et
al.(13) , we find that affinity-purified antibodies against
the adipocyte lipase recognize an
We thank Dr. Mario Ascoli, University of Iowa College
of Medicine, for generously providing the MA-10 Leydig cells, and we
are grateful for expert technical assistance by Daniel M. Levin. We are
also grateful to the following colleagues who provided experimental
results prior to publication: A. S. Greenberg, Human Nutrition Research
Center/Tufts University; T. Barber and E. J. Blanchette Mackie,
National Institutes of Health.
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
Cell Culture and Fractionation
3T3-L1 adipocyte,
Y-1 adrenal cortical cells, and MA-10 Leydig cells, all murine cell
lines, were cultured in 100-mm plates. 3T3-L1 adipoblasts were grown to
confluence and differentiated into adipocytes for 14 days(8) .
The adipocytes were prepared for Western blot analysis by lysing the
cells for 15 min in 3 ml of a lysing medium containing 10 mM Tris-HCl, pH 7.4, 1 mM EDTA, and protease inhibitors: 20
µg/ml leupeptin, 1 mM benzamidine, and 100 µM [4-(2-aminoethyl)-benzenesulfonylfluoride]
hydrochloride. Lysed cells were disrupted with 10 strokes in a
Teflon/glass homogenizer. Y-1 adrenal cortical cells (9) were
grown to confluence in Ham's F-12/Dulbecco's modified
Eagle's medium (1:1) medium containing 10% horse serum, 2.5%
fetal bovine serum, 1 mM glutamine, 10 units/ml penicillin,
and 10 µg/ml streptomycin, and maintained in a 37 °C incubator
with a 3.2% CO atmosphere. MA-10 Leydig cells (10) were grown to confluence in Waymouth's MB 752/1
medium (Life Technologies, Inc.) containing 15% horse serum, 1 mM glutamine, 10 units/ml penicillin, and 10 µg/ml streptomycin,
and maintained in a 37 °C incubator with a 3.2% CO
atmosphere. To prepare both Y-1 adrenal cortical and MA-10 Leydig
cells for Western blot analysis, the cells were washed three times with
phosphate-buffered saline, scraped from the plates in 3 ml of
phosphate-buffered saline, and pelleted by centrifugation at 300
g at room temperature for 10 min. The cells were
hypotonically lysed for 15 min in the lysing medium described above and
disrupted by 10 strokes in a Teflon/glass homogenizer; homogenates were
centrifuged at 27,000
g for 30 min at 4 °C. The
floating cholesteryl ester droplets were removed in as little volume as
possible, recentrifuged, and the supernatant under the droplets was
removed by aspiration with a fine needle. Successive centrifugations
were employed to separate the lipid droplets from the supernatant.
Homogenates of adipocytes and purified cholesteryl ester droplets from
the steroidogenic cells were extracted for 30 min at 37 °C with
2.5% SDS, 20 mM NaF, 1 mM EDTA, the protease
inhibitors as above, and 20 mM Tris-HCl, pH 7.4. After
centrifugation at 10,000
g for 15 min, the infranatant
under the undissolved lipid was removed and applied to SDS-PAGE
(
)gels (10% acrylamide and 0.2% N,N`-methylene
bisacrylamide) in Laemmli sample buffer and transferred to
nitrocellulose for Western blotting.
Immunoblotting
Polyclonal antibodies with specific
reactivity against the unique carboxyl terminus of perilipin A were
affinity-purified from rabbit antisera raised against full-length rat
perilipin A. The carboxyl-terminal peptide from perilipin A (amino
acids 421-517) was produced as a fusion protein in Escherichia coli using the QIAexpress system (QIAGEN, Inc). An
affinity column was prepared by coupling the purified peptide to
Affi-Gel® 10 resin (Bio-Rad). Antibodies were purified by
repetitive applications of rabbit antisera raised against full-length
rat perilipin A over the affinity column using a Pharmacia FPLC system.
Antibodies with specific reactivity against full-length perilipin A
were isolated from rabbit antisera raised against perilipin A purified
from fat cakes of primary rat adipocytes, as described
previously(3) .
Northern Analysis
Total RNA was extracted from rat
and murine primary adipocytes, and cultured 3T3-L1 adipocytes, Y-1
adrenal cortical cells, and MA-10 Leydig cells by the TRIzol method
(Life Technologies, Inc.). For Northern blot analysis, total RNA was
electrophoresed on 1% formaldehyde-agarose gels; the RNA was then
transferred to nitrocellulose. High stringency Northern blot analysis
using a radioactively labeled full-length perilipin A cDNA probe was
performed as described previously(4) .
57 kDa, but which migrates on
SDS-PAGE gels as a
61 kDa protein(3, 4) .
(
)Western blotting with polyclonal antibodies
affinity-purified against full-length perilipin A revealed that Y-1
adrenal cortical cells and MA-10 Leydig cells contain a protein that
co-migrates on SDS-PAGE with perilipin A from cultured 3T3-L1
adipocytes (Fig. 1A). A second major protein of
42
kDa was readily detected by the perilipin-specific antibodies in both
steroidogenic cell lines, but not in adipocytes. It is unlikely that
the 42-kDa species is a proteolytic breakdown product of perilipin A,
since the relative amounts of perilipin A and the 42-kDa protein remain
constant whether the cells are lysed immediately in a medium containing
SDS and protease inhibitors or are homogenized and subcellular
fractions are prepared. We designate the 42-kDa protein as perilipin C
(see below). There were clear quantitative differences in the amounts
of perilipins A and C in adipocytes and steroidogenic cells. When
normalized for DNA in cell homogenates, the amount of perilipin A in
isolated lipid droplets (see below) from adipocyte homogenates was at
least 20-30-fold greater than that in isolated lipid droplets
from the steroid producing cells. (
)Furthermore, prolonged
overexposure of these immunoblots that were probed with
I-protein A failed to reveal perilipin C in adipocytes
(data not shown).
Figure 1:
Western blotting of
3T3-L1 adipocytes, Y-1 adrenal cortical cells, and MA-10 Leydig cells
for perilipins. 3T3-L1 adipocytes, Y-1 adrenal cortical cells, and
MA-10 Leydig cells were cultured and processed for Western blot
analysis as described under ``Experimental Procedures.''
Western blot analyses for panelsA and B were performed with affinity-purified antibodies from antiserum
that was raised against full-length rat perilipin A. Western blot
analysis for panelC was performed with affinity-purified antibodies directed against the carboxyl
terminus of perilipin A. PanelA, Western blot of
whole adipocyte homogenates and isolated lipid droplets from Y-1
adrenal cortical and MA-10 Leydig cells. The relative amounts loaded
were equivalent to 2.5, 141, and 160 µg of DNA from 3T3-L1, Y-1,
and MA-10 cells, respectively. Panel B, Western blot
analysis of subcellular fractions of Y-1 adrenal cell homogenates: F, lipid droplets; S, supernatant; M,
membranes. A greater proportion of the total lipid droplet fraction
than of the other two fractions was loaded in order to permit viewing
of minor bands. With equivalent loads of each fraction, the amount of
perilipin in supernatant and membranes was less than 10% of that in the
lipid fraction (data not shown). By in situ immunofluorescence
microscopy, which provides far greater sensitivity than Western
blotting, the perilipin is clearly shown to be limited to the surface
of cholesteryl ester droplets (see Footnote 3). Panel
C, Western blotting of 3T3-L1 adipocyte homogenates and Y-1
adrenal lipid fractions with affinity-purified antibodies specific for
the carboxyl terminus of perilipin A. The relative loads were similar
to those for panelA.
As with adipocytes(3) , the perilipins in
the steroidogenic cells fractionated primarily with the floating lipid
droplets upon centrifugation of homogenates (Fig. 1B);
the minor amounts of perilipins A and C in supernatant and membrane
fractions from Y-1 adrenal cortical cells probably represent
contamination of these fractions with lipid droplets. Identical results were obtained upon fractionation of MA-10
Leydig cells (data not shown). In the overexposed Western blot of the
lipid fraction of the Y-1 adrenal cells (Fig. 1B), two
additional proteins were detected at
46 and
47 kDa; the
smaller protein may represent perilipin B, a minor 46-kDa species
observed in rat and murine adipocytes (4).
The larger form,
tentatively designated as perilipin D, may correspond to the 1.8-kb
mRNA expressed in both steroidogenic cells and adipocytes (see below).
Thus, perilipin A is the major species expressed in both adipocytes and
steroidogenic cells; additionally, adrenal cortical and Leydig cells
contain relatively abundant amounts of perilipin C, which is not
detected in adipocytes by Western blotting.
-terminal amino acids, but contain dissimilar COOH
termini(4) . To obtain information on the composition of
perilipin C, antibodies specific for the COOH terminus of perilipin A
were affinity-purified with the use of a polypeptide containing the
unique region of perilipin A (Fig. 1C). These antibodies
recognized perilipin A in adipocytes as well as the corresponding
protein in the adrenal cells. On the other hand, the perilipin A,
carboxyl-terminal-specific antibodies did not detect a signal at
42 kDa, suggesting that perilipin C lacks this COOH-terminal
sequence. With affinity-purified antibodies directed against
full-length perilipin A, we found that the relative amounts of
perilipins A and C differed among different isolates of Y-1 adrenal
cells. However, since we have determined that many of the epitopes
recognized by these antibodies are found in the unique COOH terminus of
perilipin A,
(
)it was not possible to quantify
the relative amounts of perilipins A and C in various cells. Finally,
the COOH-terminal-specific antibody preparation also detected a protein
of
47/48 kDa, which may represent perilipin D, a variant that is
expressed weakly in Y-1 cells relative to 3T3-L1 cells (see Figs.
1A and 2).
(
)The 3T3-L1 and
primary adipocytes also contained 3.9- and 1.8-kb mRNAs. A relatively
rare 1.5-kb species in adipocytes was detected upon overexposure of the
blots (Fig. 2, lane7). In Y-1 adrenal cortical
and MA-10 Leydig cells, the 1.5-kb mRNA was nearly as abundant as that
at 3.0 kb; relatively minor amounts of the 3.9- and 1.8-kb species were
observed. Clearly, a distinctive feature of the steroidogenic cells is
the relative abundance of the 1.5-kb mRNA. This finding parallels the
detection of the novel
42-kDa protein, suggesting that the 1.5-kb
species encodes perilipin C. Finally, the overall abundance of mRNAs
for perilipins in the steroidogenic cell lines is considerably less
than in adipocytes. Despite loading of 7 times more RNA from adrenal
and Leydig cells than from adipocytes onto the gels for Northern blot
analyses, the signal for perilipin A was weaker in the steroidogenic
cells, indicating that the relative abundance of the perilipin A mRNA
in the adrenal and Leydig cells is <5% of that in adipocytes. These
data are consistent with previous observations, which predicted that if
perilipin mRNAs were present in non-adipose tissues, they would
represent less than 0.01% of total mRNA(4) .
Figure 2:
Expression of perilipin mRNAs in primary
and cultured adipocytes and in steroidogenic cells. The Northern blot
of total RNA from primary and cultured adipocytes, Y-1 adrenal cortical
cells, and MA-10 Leydig cells was probed with full-length murine
perilipin A cDNA, which is nearly identical to rat perilipin A cDNA
(see Footnote 5). The blot contains 3 µg of total RNA from rat
primary (lane1), 3T3-L1 cultured (lanes2 and 7), and murine primary adipocytes (lane3). Lanes4 and 6 contained 20 µg of total RNA from Y-1 adrenal cortical cells,
and lane5 contained 20 µg of MA-10 Leydig cell
total RNA. Lanes 1-5 were from the same gel; lanes4 and 5 were exposed twice as long as lanes
1-3. Lanes 6 and 7 were from a different
gel that was extensively overexposed. Designations A-D indicate the perilipin variants thought to be encoded by the
different RNAs.
As in rat
adipocytes, a substantial fraction of the perilipin A in murine
adipocytes is partially phosphorylated in lipolytically quiet cells and
migrates in SDS-PAGE gels as a 62-kDa protein. Upon lipolytic
stimulation by agents that increase cellular cAMP concentrations,
perilipin A is polyphosphorylated and migrates as a 65/67 kDa rat
protein (3) or a 65-kDa murine protein. Steroidogenesis in Y-1
adrenal cortical cells is induced by ACTH or forskolin, both of which
increase cAMP concentrations(11, 12) ; treatment of
cells with either agent altered the migration of perilipin A in
SDS-PAGE gels (Fig. 3). With ACTH, approximately 25% of the
perilipin shifted upward to 65 kDa, whereas with forskolin
75% of
the protein exhibited altered migration, thus indicating greater
activation of PKA by forskolin. Colchicine, which stimulates
steroidogenesis in these cells in a non-cAMP-dependent
fashion(12) , produced no change in perilipin A behavior in
SDS-PAGE. By contrast, none of the agents produced a change in the
migration of perilipin C, which is not surprising since the PKA sites
responsible for shifting the position of perilipin A under SDS-PAGE are
in the COOH terminus(4) , the region apparently absent from
perilipin C, as noted above.
Figure 3:
Behavior in SDS-PAGE gels of perilipins A
and C from Y-1 adrenal cortical cells subjected to various
steroidogenic stimuli. Y-1 adrenal cortical cells (lanes
3-6) were incubated with no stimulating agent (Control), for
2 h with 1 µM ACTH or 10 µM forskolin, and
for 16 h with 1 µM colchicine. To terminate reactions
without reversing the effects of these agents, the cells were lysed in
the standard lysing medium (see ``Experimental Procedures'')
supplemented with 4% SDS, 6% glycerol, and 0.8% Triton X-100. The
lysates were mixed vigorously, aliquots of whole cell lysates were
subjected to SDS-PAGE, the proteins were transferred to nitrocellulose,
and the blots were probed with affinity-purified antibodies against
full-length perilipin A. For comparison, control and
isoproterenol-stimulated (10 µM) 3T3-L1 adipocyte lysates
are shown in lanes1 and 2, respectively;
the greater signal in lane2 is the result of loading
more cellular material than in lane1. In
unstimulated cells (lanes1, 3, and 6) perilipin A appears as a 61/62-kDa doublet of
dephospho- and phospho-forms, respectively; this phosphorylation is
probably not at a PKA site (3). The PKA-stimulated form is the upper
band in lanes2, 4, and 5.
Perilipins are not universal components
of intracellular lipid deposits. For example, we have failed to detect
perilipins in hepatocytes, in Ito cells of liver, and in alveolar cells
of mammary glands from lactating rats, all of which contain prominent
triacylglycerol deposits.()
Similarly, no
perilipin was detected in cholesterol-loaded J-774 macrophages, a model
for foam cells, or in lipid-laden fibroblasts from subjects with the
neutral lipid storage disease, ichthyosis.(
)
Thus, we consider the occurrence of perilipins in steroidogenic
cells to be significant, and most likely related to the similar modes
by which the cholesteryl esters of these cells and the triacylglycerols
of adipocytes are hydrolyzed.
85-kDa major and an
87-kDa
minor species in adipocytes, but only the larger species was found in
the two steroid producing cells lines (data not shown). Such data
suggest that the two hydrolases may differ, and raise the possibility
of a distinct metabolic link between the 87-kDa esterase and perilipin
C. In this regard, it will be of interest to determine the
steroidogenic capacity of cells containing different ratios of the
different species of perilipin. Finally, if perilipin serves a
functional role in the hydrolysis of lipids by the hormone-sensitive
lipase/cholesteryl esterase class of enzymes, one would expect to find
abnormal energy metabolism or steroid hormone production under
conditions of altered perilipin expression.
-terminal sequence, amino acids
17-121 (D. L. Brasaemle and C. Londos, unpublished data).
400-base pair
polymerase chain reaction-derived fragments, corresponding to the
entire murine perilipin A coding region, hybridize with the 3.0-kb mRNA
(J. Gruia-Gray, unpublished data).
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.