©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Perilipins Are Associated with Cholesteryl Ester Droplets in Steroidogenic Adrenal Cortical and Leydig Cells (*)

Diane A. Servetnick (1), Dawn L. Brasaemle (1), Jasmine Gruia-Gray (2), Alan R. Kimmel (2), J. Wolff (3), Constantine Londos (1)(§)

From the (1)Laboratory of Cellular and Developmental Biology, Membrane Regulation Section, (2)Molecular Mechanisms of Development Section and (3)Laboratory of Biochemical Pharmacology, Endocrine Biochemistry Section, NIDDK, National Institutes of Health, Bethesda, Maryland 20892

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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.


INTRODUCTION

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.


EXPERIMENTAL PROCEDURES

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) .


RESULTS AND DISCUSSION

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 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.

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-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).

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.()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.

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 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.


FOOTNOTES

*
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Bldg. 6, Rm. B1-32, National Institutes of Health, Bethesda, MD 20892-2715. Tel.: 301-496-6991; Fax: 301-496-5239; E-mail: clondos@helix.nih.gov.

The abbreviations used are: PAGE, polyacrylamide gel electrophoresis; ACTH, adrenocorticotropic hormone; PKA, cAMP-dependent protein kinase; kb, kilobase.

With minor exceptions, as noted in the present paper, the perilipin proteins found in murine adipocytes are nearly identical to those in rat adipocytes (T. Takeda, C. M. Rondinone, J. L. Theodorakis, T. Barber, E. J. Blanchette-Mackie, R. H. Pointer, A. R. Kimmel, A. S. Greenberg, and C. Londos, submitted for publication).

T. Barber, E. J. Blanchette-Mackie, J. Wolff, and C. Londos, unpublished data.

Antisera raised against perilipin that was purified from rat fat cakes have strong reactivity against a recombinant peptide containing the COOH-terminal 97 amino acids of perilipin A. These antisera have weak reactivity against recombinant peptides containing the NH-terminal sequence, amino acids 17-121 (D. L. Brasaemle and C. Londos, unpublished data).

Rat and murine perilipin A cDNAs share 93% identity at the amino acid level. Five different 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).

Blanchette-Mackie, E. J., Dwyer, N. K., Barber, T., Coxey, R. A., Takeda, T., Rondinone, C. M., Theodorakis, J. L., Greenberg, A. S., and Londos, C., J. Lipid Res. (1995) 36, in press.

A. S. Greenberg, personal communication.


ACKNOWLEDGEMENTS

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.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.