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J Biol Chem, Vol. 275, Issue 7, 5011-5015, February 18, 2000

Translocation of Hormone-sensitive Lipase and Perilipin upon Lipolytic Stimulation of Rat Adipocytes^*

Gary M. Clifford^§, Constantine Londos^¶, Fredric B. Kraemer, Richard G. Vernon^**, and Stephen J. Yeaman

From the  School of Biochemistry and Genetics, University of Newcastle, Newcastle upon Tyne NE2 4HH, United Kingdom, the ^¶ Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-2715, the  Department of Medicine, Veterans Affairs Palo Alto Health Care System, Palo Alto, California 94304, and the ^** Hannah Research Institute, Ayr KA6 5HL, United Kingdom

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Adipocyte lipolysis was compared with hormone-sensitive lipase (HSL)/perilipin subcellular distribution and perilipin phosphorylation using Western blot analysis. Under basal conditions, HSL resided predominantly in the cytosol and unphosphorylated perilipin upon the lipid droplet. Upon lipolytic stimulation of adipocytes isolated from young rats with the -adrenergic agonist, isoproterenol, HSL translocated from the cytosol to the lipid droplet, but there was no movement of perilipin from the droplet to the cytosol; however, perilipin phosphorylation was observed. By contrast, upon lipolytic stimulation and perilipin phosphorylation in cells from more mature rats, there was no HSL translocation but a significant movement of perilipin away from the lipid droplet. Adipocytes from younger rats had markedly greater rates of lipolysis than those from the older rats. Thus high rates of lipolysis require translocation of HSL to the lipid droplet and translocation of HSL and perilipin can occur independently of each other. A loss of the ability to translocate HSL to the lipid droplet probably contributes to the diminished lipolytic response to catecholamines with age.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The molecular basis of the acute hormonal regulation of lipolysis in adipocytes remains unclear. Although the rate-limiting step of lipolysis appears to be catalyzed by hormone-sensitive lipase (HSL),¹ an enzyme that is acutely regulated by hormones that elevate cAMP and activate cAMP-dependent protein kinase (reviewed in Refs. 1 and 2), the activation of purified HSL by phosphorylation in vitro (3) is insufficient to account for the stimulation of lipolysis in vivo (4), suggesting the involvement of additional mechanisms.

It has now been shown that upon lipolytic stimulation of the fat cell, HSL protein translocates from a cytoplasmic compartment to the lipid droplet (5), and subsequent immunofluorescence studies using anti-HSL antibodies in 3T3-L1 adipocytes have demonstrated that HSL is distributed throughout the cytoplasm of unstimulated cells, but moves to the surface of lipid droplets in these cells upon lipolytic stimulation (6). In addition to the well established mechanism of cAMP-dependent activation of HSL, Okuda et al. (7, 8) proposed the so-called "hormone-sensitive substrate theory" of lipolysis in which the hormone did not act on the lipase, but on the endogenous lipid substrate. Wise and Jungas (9) went on to propose a dual mechanism of lipolytic activation by catecholamines involving "substrate activation," suggesting that some factor at the surface of intact lipid droplets may be necessary for the hormonal stimulation of lipolysis. More recent work would predict that this factor would be expected to facilitate the translocation of HSL, presumably by some alteration in the lipid droplet surface, and may involve the formation of smaller lipid droplets observed in 3T3-L1 adipocytes and Leydig cells (10, 11).

Recent studies have demonstrated that differences in patterns of lipid droplet protein expression are partly responsible for the differing lipolytic response to catecholamines observed in adipocytes from different fat depots (12), suggesting the involvement of one or more protein factors. One candidate protein for this role is perilipin, the predominant phosphoprotein in adipocytes. Perilipin is located at the lipid droplet surface, the presumed site of HSL action upon translocation to its triacylglycerol substrate (13). Perilipin has been detected primarily in adipocytes and steroidogenic cells, in which lipid droplet hydrolysis is stimulated by cyclic AMP and mediated by HSL (14). Furthermore perilipin and HSL are concomitantly phosphorylated in response to lipolytic hormones in intact adipocytes and both are dephosphorylated in the presence of insulin (15, 16), although different phosphatases may be involved (17). Recently, tumor necrosis factor  was shown to increase lipolysis in 3T3-L1 adipocytes by a mechanism that involved a reduction in perilipin expression and also a redistribution of perilipin protein in the cell (18). Furthermore overexpression of perilipins A and B in 3T3-L1 adipocytes blocked the ability of tumor necrosis factor , but not isoproterenol (a -adrenergic agonist), to increase lipolysis (19).

Thus it has been proposed that in nonstimulated cells perilipin may deter HSL interaction with the lipid droplet by forming a barrier around it, whereas cAMP-dependent protein kinase-phosphorylated perilipin may somehow allow access of the enzyme to the droplet, perhaps by a modification of its surface (6, 18). In the present study, we undertake the characterization of adipocyte HSL/perilipin translocation and the phosphorylation of perilipin in response to isoproterenol. We demonstrate that the stimulation of lipopolysis in adipocytes is closely paralleled by perilipin phosphorylation and that depending on the age of the rats, there is either translocation of HSL toward or perilipin away from the lipid droplet. Translocation of HSL to the droplet is associated with the greater lipolytic rates seen in young rats.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials-- [³²P]Orthophosphate was purchased from ICN. The protease inhibitors pepstatin, leupeptin, and antipain were from the Peptide Institute, Osaka, Japan. Collagenase was from Worthington, and orlistat (also called Zenical or tetrahydrolipstatin) was a kind gift from Hoffmann LaRoche, Basel, Switzerland. Anti-perilipin NH₂-terminal antibody was raised in rabbits as described previously (14), as was an antibody raised against rat HSL/bacterial fusion protein (20). All other reagents were obtained from Sigma (Poole, Dorset, United Kingdom). Male Wistar rats, >180 g, fed ad libitum on a diet of standard laboratory chow, were raised in house at the Comparative Biology Center at the University of Newcastle upon Tyne. Rats were used when they were either 6-8 weeks old (180-220 g body weight) or at 8-12 weeks (230-280 g body weight).

Adipocyte Isolation-- Adipocytes were isolated by collagenase digestion of the epididymal fat pads (21) of male Wistar rats, starved overnight, and killed by cervical dislocation. Manipulations of adipocytes for ³²P labeling were performed in reduced phosphate (50 µM KH₂PO₄) Krebs-Ringer, buffered with 25 mM Hepes, pH 7.4, containing 2.5 mM CaCl₂, 2.5 mM MgCl₂, 3% bovine serum albumin, and 2 mM glucose (reduced phosphate KRH). 200 nM adenosine was included to suppress cAMP production and stimulation of cAMP-dependent protein kinase activity (22). All other manipulations of adipocytes were performed in standard Krebs-Ringer solution containing similar additions. Following isolation, cells were shaken at 37 °C for 1 h. Cells were then washed in bovine serum albumin-free buffer supplemented with 200 nM adenosine. The packed cell volume (PCV) of the final suspension was determined by aspirating small aliquots into capillary hematocrit tubes and centrifugation in a microhematocrit centrifuge.

Preparation of Cytosolic and Fatcake Fractions for SDS-PAGE-- Aliquots (300 µl) of adipocytes at approximately 20% PCV were incubated for the indicated times in 2 ml wells of 48-well tissue culture plates at 37 °C, either under "basal" conditions, i.e. supplemented with 200 nM adenosine and 2 mM glucose only, or in the presence of the additions mentioned in the text. KRH buffer (150 µl) was then removed from below the floating cells for the assay of glycerol release, and the remaining cells lysed in 150 µl of ice-cold 50 mM Tris-HCl buffer, pH 7.4, containing 225 mM sucrose, 1 mM EDTA, 1 mM benzamidine, 1 µg/ml pepstatin, 1 µg/ml leupeptin, 1 µg/ml antipain, and 50 mM NaF (buffer A). Following lysis, cells remained on ice for 15 min for the floating fatcake to solidify. The lysate was then vortexed vigorously and centrifuged at 13,000 × g at 4 °C for 15 min. The cytosolic fraction was aspirated from below the solidified fatcake and 100 µl of cytosol added to an equal volume of 2× sample buffer for SDS-PAGE. The fatcake fraction was respun at 13,000 × g at 4 °C for 15 min and any contaminating cytosol aspirated and discarded. The fatcake was warmed to room temperature, 100 µl of SDS sample buffer added, and the solution vortexed thoroughly. Following centrifugation at 13,000 × g at 4 °C for 15 min, the fatcake protein extract was aspirated from below the floating fat layer for PAGE. Dilution factors and packed cell volumes from each experiment were taken into consideration to ensure equivalent loading (per ml of packed cells) of the two fractions on subsequent SDS-PAGE.

Preparation of ³²P-Labeled Adipocyte Extracts-- Adipocytes (200 µl PCV/ml) were loaded with [³²P]P_i, by incubating the cells in reduced phosphate KRH (bovine serum albumin-free) supplemented with 200 nM adenosine and 200 µCi/ml [³²P]orthophosphate for 1 h at 37 °C. ³²P-Labeled adipocytes were either incubated under "basal" conditions or lipolytically stimulated with the indicated concentration of isoproterenol for the required time. Cells were allowed to float to the surface, and the infranatant was aspirated. The remaining adipocytes were lysed with ice-cold buffer A containing 3% (v/v) Triton N-101. The lysate was then vortexed vigorously and centrifuged at 13,000 × g for 5 min at 4 °C. Solubilized protein extract was aspirated from under the solidified lipid fraction and diluted with an equal volume of 2× sample buffer for PAGE.

SDS-PAGE and Western Blot Analysis-- SDS-PAGE was performed using a Tris/glycine buffer system with Hoeffer mini-gel apparatus (23). For Western blotting, protein samples subjected to SDS-PAGE (10% gels) were transferred onto polyvinylidine difluoride membranes, probed with anti-HSL or anti-perilipin antibodies, and the amount of immunoreactive protein determined by enhanced chemiluminescence reagents from Amersham, Bucks, UK. Phosphoproteins were visualized by exposure to Fujifilm.

Estimation of Glycerol Release-- Aliquots (20 µl) were withdrawn from medium of cells at 200 µl cells/ml PCV and added to 200 µl GPO-Trinder reagent (Sigma Diagnostics). This procedure was a modification of the method described in Ref. 24. Glycerol release is expressed as nanomoles glycerol ml¹ PCV min¹.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

HSL Translocation upon Lipolytic Stimulation of Adipocytes-- The subcellular location of HSL was studied in adipocytes in the absence and presence of catecholamine to investigate its relationship with the stimulation of lipolysis. Cells were isolated, incubated with increasing concentrations of isoproterenol, and the infranatant and fatcake fractions of cells analyzed for HSL content by Western blotting of SDS-PAGE separated proteins (Fig. 1A). Upon incubation with isoproterenol of adipocytes isolated from the epididymal fat pads of young Wistar rats weighing between 180 and 220 g, HSL was observed to translocate from the cytosolic fraction to the fatcake fraction in a dose-responsive manner (Fig. 1B). Under basal conditions, approximately 40% of the immunoreactive HSL was recovered in the fatcake fraction. Using 1 µM isoproterenol to achieve maximal lipolysis (mean glycerol release = 170 nmol/ml cells/min), approximately 40% of the total HSL translocated to the fatcake fraction, with 80% of total HSL residing in the fatcake fraction after 5 min of stimulation. This isoproterenol-induced HSL translocation event correlated with stimulation of lipolysis, as estimated by glycerol release from the adipocytes (Fig. 1B). Furthermore, in a separate set of experiments in which 75% of the HSL was cytosolic under basal conditions, most of the HSL translocation in response to 1 µM isoproterenol, i.e. 50% of HSL translocating from cytosol to fatcake, occurred within the first 2 min of lipolytic stimulation, with the stimulation of lipolysis occurring over a similar time frame (Fig. 2). Correlation analysis of results from these and other experiments with young male rates (180-220 g body weight) showed a highly significant (p < 0.001) linear correlation between the rate of lipolysis and the proportion of HSL associated with the fatcake (not shown). Incubation of cells with orlistat, an inhibitor of HSL (25), inhibited maximally stimulated lipolysis by up to 50% without any significant effect on HSL translocation, suggesting that the translocation is not dependent on the catalytic function of HSL.²

View larger version (48K): [in this window] [in a new window]   Fig. 1.   Isoproterenol-stimulated translocation of HSL. Epididymal adipocytes from young male Wistar rats (180-220 g) were maintained under basal conditions (Bas) or stimulated with the indicated concentration of isoproterenol. After 5 min, samples were removed for measurement of glycerol release, and the cells were then lysed and fatcake and cytosolic fractions prepared. Fractions were subjected to SDS-PAGE and Western blotted with anti-HSL antibodies (A). Densitometric analysis allowed a quantitative measurement of the localization of HSL (B). Data represent the mean ± S.E., n = 14. *, p < 0.01 versus basal; **, p < 0.005 versus basal.

View larger version (27K): [in this window] [in a new window]   Fig. 2.   Time course of isoproterenol-stimulated lipolysis and HSL translocation. Epididymal adipocytes from young male rats (180-220 g) were maintained under basal conditions or lipolytically stimulated in the presence of 1 µM isoproterenol. After the times indicated, samples were removed for measurement of glycerol release, the cells were lysed, and fatcake and cytosolic fractions prepared. Fractions were then subjected to SDS-PAGE and Western blotted with anti-HSL antibodies. Densitometric analysis allowed a quantitative measurement of the localization of HSL. Data represent the mean ± S.E., n = 4. *, p < 0.05 versus basal; **, p < 0.005 versus basal.

In contrast to the above, studies with adipocytes from older rats (230-280 g body weight) showed no statistically significant translocation of HSL to the lipid droplet in response to isoproterenol, with approximately 80% of HSL being in the cytosol in both stimulated and unstimulated cells (Fig. 3B). Isoproterenol still stimulated lipolysis in these cells from older rats, but to a lesser extent (46 nmol/ml cells/min), than in cells from younger animals (170 nmol/ml cells/min) (Fig. 3).

View larger version (26K): [in this window] [in a new window]   Fig. 3.   HSL and perilipin translocation upon lipolytic stimulation in adipocytes from young and mature male Wistar rats. Epididymal adipocytes were used from young (180-220 g) (A) or mature (230-280 g) (B) male Wistar rats. Cells were maintained under basal conditions or stimulated with indicated concentration of isoproterenol. After incubation for 5 min, the cells were lysed, fatcake and cytosolic fractions prepared, and samples subjected to SDS-PAGE and Western blotted with anti-HSL and anti-PAT antibodies. Densitometric analysis allowed a quantitative measurement of the localization of HSL and perilipin. Data represent the mean ± S.E.; *, p < 0.05 versus basal; **, p < 0.005 versus basal. In A, n = 14 for glycerol release and HSL distribution, n = 3 for perilipin distribution. In B, n = 6 for all values. C shows a representative Western blot of perilipin translocation in mature rats.

Perilipin Phosphorylation upon Lipolytic Stimulation-- Consistent with previous reports (13, 15), anti-perilipin NH₂-terminal (anti-PAT) antibodies used in this study to identify perilipin were immunoreactive against a 65-kDa protein in adipocytes, corresponding to the predominant 65-kDa phosphoprotein in ³²P-loaded adipocyte extracts (Fig. 4), hereby referred to as perilipin. This corresponds to the polypeptide of estimated mass 62 kDa reported using slightly different electrophoretic conditions (13).

View larger version (86K): [in this window] [in a new window]   Fig. 4.   Identification of 65-kDa adipocyte phosphoprotein as perilipin. Fatcake fraction from basal cells was analyzed by SDS-PAGE. Protein molecular weight markers (A), Western blot of extract from adipocytes probed with anti-PAT antibodies (B), and phosphorimage of 32P-labeled adipocyte fatcake fraction (C) are shown.

Incubation of adipocytes from young rats (180-220 g) with increasing concentrations of isoproterenol was accompanied by an increase in the phosphorylation of perilipin in the extracts. This dose-responsive phosphorylation of perilipin was observable both by phosphoprotein analysis of perilipin in ³²P-loaded extracts (Fig. 5A) and by a decrease in electrophoretic mobility from the 65-kDa perilipin band to a 65/67-kDa doublet upon Western blot analysis with anti-PAT antibodies (Fig. 5B). Perilipin phosphorylation occurred in a manner closely paralleling the dose-responsive stimulation of lipolysis in these cells (Fig. 1), with near-maximal phosphorylation being achieved with 100 nM isoproterenol.

View larger version (79K): [in this window] [in a new window]   Fig. 5.   Isoproterenol-stimulated phosphorylation of perilipin. Epididymal adipocytes from young male Wistar rats were loaded with 32P and maintained under basal conditions or stimulated with the indicated concentration of isoproterenol. After 5 min, whole extracts were prepared, then subjected to SDS-PAGE and phosphorimaged (A). Non-32P-loaded adipocytes isolated under similar conditions were also stimulated with similar concentrations of isoproterenol for 5 min (B), or incubated with 1 µM isoproterenol for the length of time indicated (C), were then lysed and fatcake and cytosolic fractions prepared. Fractions were subjected to SDS-PAGE and Western blotted with anti-PAT antibodies.

³²P-Loaded adipocytes were incubated with 1 µM isoproterenol to elicit a maximal lipolytic response, and samples were removed from incubations at various times after lipolytic stimulation for SDS-PAGE and phosphorimage analysis. Maximal phosphorylation of perilipin occurred almost completely within the first 2 min of incubation with isoproterenol (Fig. 5C), again paralleling the stimulation of lipolysis in these cells (Fig. 2).

Thus, upon incubation with isoproterenol, the observed phosphorylation and shift in electrophoretic mobility of perilipin parallels the stimulation of lipolysis in both a time- and dose-dependent manner.

Perilipin Translocation upon Lipolytic Stimulation in Adipocytes-- To determine the subcellular localization of perilipin upon lipolytic stimulation, the distribution of perilipin was studied under different conditions. Under basal conditions perilipin was predominantly associated with the fatcake fraction from all cells studied. In cells isolated from young male rats (in which HSL translocates significantly upon lipolytic stimulation), perilipin remained tightly associated with the fatcake under all conditions studied. Even when cells were incubated with 1 µM isoproterenol for 5 min to give a maximal stimulation of lipolysis and a lipolytic rate 5-fold greater than under basal conditions, there was no significant alteration in the subcellular distribution of perilipin, with approximately 90% of the total perilipin remaining fatcake-associated (Fig. 3A). Perilipin was undergoing multiple phosphorylation upon lipolytic stimulation in these cells as shown by the decrease in electrophoretic mobility (Fig. 5C).

In contrast, in cells isolated from older male rats (230-280 g body weight) there was a significant redistribution of perilipin observable upon lipolytic stimulation (Fig. 3B). Upon stimulation of these cells with increasing concentrations of isoproterenol, perilipin significantly translocated away from the fatcake fraction and into the cytosol in a dose-responsive manner (p < 0.05), with approximately 50% of the total perilipin relocating to the cytosol upon maximal lipolytic stimulation.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

This study demonstrates that the translocation of HSL in response to the -adrenergic agonist isoproterenol closely parallels the stimulation of lipolysis in young male rats. Whereas HSL remained predominantly cytosolic in adipocytes under conditions of basal lipolysis, up to 80% of the total HSL protein localized with the lipid droplet upon lipolytic stimulation. Translocation of HSL in response to an adrenergic stimulus has been demonstrated previously in young rats (180-200 g) (5). By contrast, with adipocytes from older rats, no translocation of HSL was apparent, and furthermore, the maximum rate of lipolysis induced by isoproterenol was markedly lower than in cells from younger rats. Results for older rats emphasize that lipolytic stimulation is not due to HSL translocation alone.

This study suggests that within the intact adipocyte, the phosphorylation state of perilipin at the lipid droplet surface, observable both by ³²P-incorporation as in Ref. 15, and its shift in electrophoretic mobility, is under tight control by lipolytic hormones. If perilipin is involved in the regulation of lipolysis, it may constitute the factor that localizes HSL to its substrate. Although it has been proposed that the function of perilipin could be to directly anchor HSL to its substrate as a "docking" protein when phosphorylated (5), the present work suggests that this is not the case, as within the relatively nonlipolytically responsive adipocytes from older rats, although fatcake-associated perilipin is multiply phosphorylated upon lipolytic stimulation, there is no significant translocation of HSL to the lipid droplet. Furthermore, attempts in this laboratory to co-immunoprecipitate HSL and perilipin offer no evidence of any interaction between these two proteins (not shown), nor does a yeast two-hybrid screen (26).³

Western blotting of adipocyte subcellular fractions with anti-perilipin antibodies show that under certain conditions, perilipin moves away from its location on the lipid droplet into the cytosolic fraction upon lipolytic stimulation, supporting a recent report demonstrating a similar redistribution of perilipin in 3T3-L1 adipocytes (19, 27). This translocation is unlikely to involve a conformational change that allows perilipin to become freely soluble in the cytosol as it is highly hydrophobic (13). Rather it seems more likely that the phosphorylation of perilipin and/or some other mechanism is causing an alteration in the lipid droplet surface. Indeed, in 3T3-L1 adipocytes (10) and Leydig cells (28), lipolytic stimulation is associated with the formation of many small lipid droplets from the surface of larger ones, and perilipin may be present on the surface of these smaller lipid droplets. Alternatively, the phosphorylation of perilipin or some other factor may cause an alteration at the lipid droplet surface, causing them to become more susceptible to disruption by fractionation procedures, resulting in the appearance of perilipin in the cytosolic fraction. However, it appears that the translocation of perilipin is not essential for the stimulation of lipolysis in all cells, as it is not observable in adipocytes from the young male rats in which translocation of HSL was apparent on stimulation with isoproterenol and which had the highest lipolytic rates.

A decrease in catecholamine-induced lipolysis in rat adipocytes with age observed in this study has been previously reported (29, 30). Furthermore, fasting for 24 h caused a much greater loss of adipocyte lipid in 6-week-old rats than in rats of 8-12 weeks of age (31). The mechanisms responsible for the decrease in lipolytic response of adipocytes with age have not been fully resolved, but do not appear to be due to changes in the ability to activate adenylate cyclase or to generate cAMP, suggesting a change downstream of cAMP-dependent protein kinase (32). A loss of the ability to translocate HSL to the lipid droplet may thus contribute to this diminished response to a lipolytic challenge. It is interesting to speculate that this may be due to changes at the surface of the lipid droplet, such as the loss of vimentin observed during droplet growth in 3T3-1 adipocytes (33). Similarly, as rats mature there may be changes in the levels of proteins which interact with HSL such as adipocyte lipid-binding protein (26), or lipotransin, which may localize HSL to the lipid droplet in a hormone-sensitive manner (34).

	FOOTNOTES

^* This work was supported in part by National Institutes of Health Grants DK46942 and DK49705 and by the Research Service of the Department of Veterans Affairs.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ Recipient of postgraduate studentship from the Biotechnology & Biological Sciences Research Council, United Kingdom.

To whom correspondence should be addressed: School of Biochemistry and Genetics, The Medical School, University of Newcastle, Newcastle upon Tyne NE2 4HH, UK. Tel.: 44-191-222-7433; Fax: 44-191-222-7424; E-mail: s.j.yeaman@ncl.ac.uk.

² G. M. Clifford and S. J. Yeaman, unpublished results.

³ C. J. Schultz and C. Londos, unpublished results.

	ABBREVIATIONS

The abbreviations used are: HSL, hormone-sensitive lipase; PAGE, polyacrylamide gel electrophoresis; PCV, packed cell volume; KRH, Krebs-Ringer Hepes buffer; PAT, perilipin NH₂-terminal.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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