Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes

Biochimica et Biophysica Acta 1761 (2006) 247 – 255 http://www.elsevier.com/locate/bba

Calyculin and okadaic acid promote perilipin phosphorylation and increase lipolysis in primary rat adipocytes Jinhan He a,b , Hongfeng Jiang a,b , John T. Tansey c , Chaoshu Tang a,b , Shenshen Pu a,b , Guoheng Xu a,b,⁎ a

Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100083, China Key Laboratory of Molecular Cardiovascular Sciences, Ministry of Education of China, Beijing 100083, China c Chemistry Department, Otterbein College, Westerville, OH 43081-2006, USA

b

Received 28 November 2005; received in revised form 20 January 2006; accepted 2 February 2006 Available online 28 February 2006

Abstract Lipolysis is primarily regulated by protein kinase A (PKA), which phosphorylates perilipin and hormone-sensitive lipase (HSL), and causes translocation of HSL from cytosol to lipid droplets in adipocytes. Perilipin coats lipid droplet surface and assumes to prevent lipase access to triacylglycerols, thus inhibiting basal lipolysis; phosphorylated perilipin facilitates lipolysis on PKA activation. Here, we induced lipolysis in primary rat adipocytes by inhibiting protein serine/threonine phosphatase with specific inhibitors, okadaic acid and calyculin. The incubation with calyculin promotes incorporation of 32Pi into perilipins, thus, confirming that perilipin is hyperphosphorylated. The lipolysis response to calyculin is gradually accompanied by increased accumulation of phosphorylated perilipin A in a concentration- and time-responsive manner. When perilipin phosphorylation is abrogated by the addition of N-ethylmaleimide, lipolysis ceases. Different from a considerable translocation of HSL upon PKA activation with isoproterenol, calyculin does not alter HSL redistribution in primary or differentiated adipocytes, as confirmed by both immunostaining and immunoblotting. Thus, we suggest that inhibition of the phosphatase by calyculin activates lipolysis via promoting perilipin phosphorylation rather than eliciting HSL translocation in adipocytes. Further, we show that when the endogenous phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin to the hyperphosphorylated species, and induces enhanced lipolysis. Apparently, as PKA phosphorylates perilipin and stimulates lipolysis, the phosphatase acts to dephosphorylate perilipin and attenuate lipolysis. This suggests a two-step strategy governed by a kinase and a phosphatase to modulate the steady state of perilipin phosphorylation and hence the lipolysis response to hormonal stimulation. © 2006 Elsevier B.V. All rights reserved. Keywords: Lipolysis; Perilipin; Okadaic acid; Calyculin; Phosphorylation; Dephosphorylation; Protein phosphatase; Hormone-sensitive lipase; Adipose triglyceride lipase; Lipid droplet; Isoproterenol

1. Introduction

Abbreviations: AGTL, Adipose triglyceride lipase; fDMEM, Phenol redfree and serum-free Dulbecco's modified Eagle's medium; HSL, Hormonesensitive lipase; PCV, Packed cell volume; PKA, cAMP-dependent protein kinase A; PPase, Protein serine/threonine phosphatase; PP1, protein phosphatase-1; PP2A, Protein phosphatase-2A; SDS-PAGE, SDS polyacrylamide gel electrophoresis ⁎ Corresponding author. Department of Physiology and Pathophysiology, Peking University Health Science Center, Beijing 100083, China. Tel./fax: +86 10 8280 2916. E-mail address: [email protected] (G. Xu). 1388-1981/$ - see front matter © 2006 Elsevier B.V. All rights reserved. doi:10.1016/j.bbalip.2006.02.001

Adipocyte lipolysis is the lipase-catalyzed hydrolysis of triacylglycerol to glycerol and free fatty acids, which is physiologically stimulated by catecholamines through elevating cAMP and activating cAMP-dependent protein kinase A (PKA) [1,2]. PKA phosphorylates hormone-sensitive lipase (HSL) and causes translocation of HSL from the cytosol to the lipid droplets in adipocytes [3–5], an action that enhances triacylglycerol breakdown [1,2]. Although important, HSL is not the only lipase for triacylglycerol hydrolysis because adipocytes derived from HSL null mice retain ~ 50% of residual lipase activity and are responsive to adrenergic lipolytic stimulation [6]. An adipose

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triglyceride lipase (ATGL) was recently identified to be abundant on the cytosol and the lipid droplets in adipocytes and is predominantly responsible for the initial step of triacylglycerol hydrolysis [7,8]. Besides the catalytic lipases, perilipins are a family of structural proteins that coat the surface of lipid droplets in adipocytes [9,10] and are proposed to modulate lipolysis as triacylglycerol substrate-associated regulator [1,2,5]. Perilipin A is the major isoform and perilipin B is less abundant and a shorter variant. Both proteins arise from alternatively spliced transcripts and share a common N-terminal domain of 1 to 405 amino acids [11,12]. A lower level of adipocyte perilipin is associated with higher lipolytic activity and higher circulating concentrations of free fatty acids in human obese individuals [13]. The perilipin deficiency leads to an increased level of basal lipolysis but an attenuated response to β-adrenergic-stimulated lipolysis in adipocytes derived from perilipin-null mice [14]. By contrast, the expression of ectopic perilipin A in fibroblasts inhibits basal lipolysis in the absence of PKA stimulation [5,15–17]. Upon PKA activation, perilipin A is phosphorylated at up to six serine residues [1,2]. This phosphorylation ensures lipolysis mediated by either HSL [5,15,17] or non-HSL lipase [5,15,16] in the reconstituted fibroblasts. Further, a recent study revealed that the phosphorylation of perilipin A is required for the translocation of HSL to the lipid droplets during PKA-stimulated lipolysis [5]. These observations suggest that perilipin A functions as both a suppressor of basal lipolysis and a necessary enhancer of PKAstimulated lipolysis [1,2]. Though the control of lipolysis has been long focused on cAMP and cAMP-regulated protein kinase, PKA, the results of three studies implied that protein serine/threonine phosphatase (PPase) might also participate in the lipolytic cascade [18–20]. A 1997 study provided the first clue that adipocyte lipolysis can be stimulated by specific PPase-1 (PP1) and -2A (PP2A) inhibitors, okadaic acid and calyculin [18]. Later, in adipocytes and the cellfree system consisting of isolated endogenous lipid droplets and HSL, okadaic acid was found to stimulate lipolysis, an action that did not result from increased activity of HSL but was attributable in part to a translocation of HSL and a change in the surface physicochemical character of lipid droplets [19]. However, this finding was not directly examined. As well, okadaic acid was found to inhibit perilipin dephosphorylation in vitro in adipocyte extracts and increase lipolysis in adipocytes [20]. Nonetheless, the molecular basis by which inhibition of PPase activates lipolytic response in intact fat cells has still not been resolved. In this study, we induced lipolysis by PPase inhibition with calyculin and okadaic acid in primary rat adipocytes. Incubation with calyculin promoted the incorporation of 32Pi into perilipins, thus confirming that perilipin is hyperphosphorylated. We show that lipolysis activated by calyculin is gradually accompanied by an accumulation of phosphorylated perilipin A but is not a consequence of accelerated redistribution of HSL from the cytosol to the lipid droplets in adipocytes. As perilipin phosphorylation is abrogated by the addition of Nethylmaleimide, lipolysis also ceases, thus, suggesting that the phosphorylation of perilipin rather than the translocation of HSL plays an important role for the lipolytic action on

inhibition of the PPase with calyculin. Further, we show that when the phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin A to the hyperphosphorylated species, therefore resulting in an enhanced lipolysis. These data suggest that the endogenous phosphatase participates in the lipolytic regulation in cooperation with PKA by acting to modulate the steady state of the phosphorylation perilipin A. 2. Materials and methods 2.1. Materials Calyculin A was purchased from Cell Signaling Technology (Beverly, MA). Okadaic acid, N-ethylmaleimide, phenol red-free Dulbecco's modified Eagle's medium (DMEM) containing 5 mM glucose, and horseradish peroxidase conjugated lgG, were from Sigma (St. Louis, MO). Polyclonal antibodies against rat perilipin [21] or rat HSL [4] were generous gifts from C. Londos at the U.S. National Institutes of Health. Nitrocellulose membrane, prestained protein molecular weight markers, and SuperEnhanced chemiluminescence detection reagents were from Applygen Technologies Inc. (Beijing, China).

2.2. Isolation and culture of primary rat adipocytes Adipocytes were isolated from epididymal fat pads of Sprague–Dawley rats (160–200 g) [22]. The fat pads were minced and digested in 5 ml Krebs–Ringer solution containing 25 mM HEPES, pH 7.4, 1 mg/ml type I collagenase and 1% defatted bovine serum albumin (BSA). After incubation for 40 min at 37 °C in a water bath shaken at 100 cycles/min, cells were filtered through a nylon mesh and washed 3 times in pre-warmed phenol red-free and serum-free DMEM (fDMEM). Adipocytes floating in the tube were centrifuged at 200×g for 3 min. Packed adipocytes were diluted in 1% BSA-fDMEM to generate a 10% (v/v) cell suspension. The packed cell volume (PCV) of the final suspension was determined according to the reported method [23]. Prior to the treatment, the fat cells were incubated for 1 h at 37 °C and shaken at 30 cycles/min, to restore intracellular cAMP level to a basal level. We observed that incubation of adipocytes in fDMEM but not in Krebs–Ringer solution reduced variability of cell and basal lipolysis.

2.3. Lipolysis assays A total of 50 μl of packed adipocytes was suspended in 500 μl fDMEM (10% PCV) and treated as described. The culture medium was collected and heated at 70 °C for 10 min, to inactivate residual lipases. Glycerol released in the medium served as an index of lipolysis and was determined by use of a colorimetric assay (GPO Trinder reaction) from the absorption at 490 nm [24]. Lipolysis data were expressed as micromolecules of glycerol released per milliliter PCV.

2.4. Preparation of cytosolic and fat cake fractions from primary rat adipocytes Following treatment, the culture media was assayed for glycerol. The fat cells were packed by centrifuging the cells for 3 min at 200×g, and homogenized in ice-cold fractionation buffer (50 mM Tris–HCl, pH 7.4, 255 mM sucrose, 1 mM EDTA, 0.1 mM sodium orthovanadate [Na3VO4], and 50 mM sodium fluoride [NaF]), as described [3,25]. The cell lysate was incubated on ice for 15 min and then centrifuged at 20,000×g for 30 min at 4 °C. The solidified fat cake of intracellular lipid droplets floated on top of the tube. The cytosolic fraction was localized below the layer of the fat cake. The fractions were collected, mixed with concentrated Laemmli sample buffer (62 mM Tris–HCl, pH 6.8, 5% SDS, 1% beta-mercaptoethenol, 0.1 mM Na3VO4, 50 mM NaF, and 15% glycerol in final) and adjusted equivalently against PCV [3]. The samples were heated to 95 °C for 5 min and cleaned at 12,000×g for 10 min, prior to loading on SDS-PAGE.

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2.5. Immunoblotting Protein content of packed adipocytes was determined by use of a bicinchoninic acid protein assay kit (Pierce). Incubation for 1 h with calyculin or other agents did not alter the total protein content of adipocytes in individual treatments. Thus, the protein loading for SDS-PAGE was equivalently adjusted against PCV. For immunoblotting of perilipin, equal amounts of fat cake extracts were loaded and separated by SDS-PAGE on a low bis concentration gel (10% acrylamide and 0.07% N,N′-methylene-bis-acrylamide), which provides better resolution of proteins in the 60- to 70-kDa range [9]. For immunoblotting of HSL, equivalent amounts of protein extracted from the cytosolic and fat cake fractions were separated by 8% SDS-PAGE. After electrophoresis, the proteins were transferred to nitrocellulose membranes and immunoblotted as described [26,27]. Briefly, the membranes were blocked with 5% non-fat milk in TBS-T buffer (150 mM NaCl, 20 mM Tris–HCl, pH 7.4, 0.05% Tween-20), and incubated at room temperature for 1 h with antibodies against rat perilipin, or against rat HSL, respectively. After being washed for 3 × 10 min in TBS-T buffer, the membranes were probed for 1 h with secondary antibodies conjugated to horseradish peroxidase. The blots were washed and then developed by use of an enhanced chemiluminescent detection method [11]. The protein bands were visualized after exposure of the membranes to Kodak X-ray film.

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cubation with 5 nM calyculin induces a slight increase (1.3-fold) in lipolysis (Fig. 1A), which was accompanied by a minimal phosphorylation of perilipin A (Fig. 1B). As the concentration of calyculin was increased to 10, 15, or 20 nM, lipolysis was gradually increased by 2.6-, 6.4-, or 12.2-fold, respectively, and significant accumulation of phosphorylated perilipin A occurred (Fig. 1A, B). A 40-nM concentration of calyculin resulted in a dispersal of phosphorylated perilipin A, seen as multiple bands in the gel (Fig. 1B). This maximally promoted lipolysis by 16.9-fold (Fig. 1A). The median effective

2.6. 32P-loading of adipocytes and autoradiography of fat cake extracts Adipocytes (10% PCV) were loaded with 100 μCi/ml [32P]-orthophosphate for 90 min at 37 °C. 32P-loaded fat cells were incubated for an additional 30 min in the absence or presence of isoproterenol or 20 nM calyculin in Krebs–Ringer solution with reduced (50 μM) phosphate and 1% defatted bovine serum albumin [9]. The cells were collected and immediately homogenized. The fractionation and extraction of fat cakes were performed as described above. The proteins were separated on 10% SDS-PAGE and bands were visualized by autoradiography. A parallel experiment was performed under the same conditions as with the absence of 32Pi in the incubation medium, and proteins extracted from the fat cake were subjected to immunoblotting with an anti-perilipin antibody.

2.7. Differentiation of rat preadipocytes and immunostaining of HSL Adipose precursor cells were isolated from epididymal fat pads of 6-weekold Sprague–Dawley rats (~150 g), and differentiated into adipocytes for 3 days in serum-free DMEM-F12 (1:1) medium supplemented with 5 μg/ml insulin, 33 μM biotin, and 200 pM triiodothyronine [28]. The differentiated adipocytes (day 5) were treated with 10 μM isoproterenol for 5 min or with 20 nM calyculin for 5, 15, 20, and 30 min. Immunostaining was performed according to the prior methods [5,27]. The cells were fixed for 20 min with 4% paraformaldehyde and 0.01% Triton X-100 in PBS buffer at room temperature followed by 3 rinses for 5 min each with PBS. Nonspecific binding sites in the cells were blocked with 2% donkey serum for 60 min and washed 3 times for 5 min each. The cells were incubated with rabbit polyclonal serum against HSL at 1:200 overnight at 4 °C and subsequently with FITC-conjugated donkey anti-rabbit lgG at 1:200 for 1 h in the dark at room temperature. Immunofluorescent signal of HSL was observed with use of a Nikon Eclipse TE2000-U microscope.

2.8. Statistical analysis Data are expressed as means ± S.E. One-way ANOVA Tukey's test was performed for statistical analysis with use of GraphPad Prism version 4.0. A P value < 0.05 was considered statistically significant.

3. Results 3.1. Calyculin increases lipolysis and phosphorylation of perilipin A in a concentration- and time-responsive manner Calyculin A specifically inhibit PPase activity in primary rat adipocytes [29] and in nonadipose cells [30,31]. A 1-h in-

Fig. 1. Increase in lipolysis is accompanied by phosphorylation of perilipin A in a concentration- and time-dependent manner. Adipocytes were incubated with calyculin for 1 h or indicated period. (A and C) lipolysis was stimulated by calyculin in a concentration- (A) and time- (C) dependent manner. Lipolysis data are the means ± S.E. of 5 separate experiments, each performed in triplicate; white circles, incubation for 1 h with calyculin at different concentrations; black squares, calyculin; black triangles, vehicle control. (B and D) proteins extracted from the fat cake fraction of rat adipocytes were immunoblotted with an antiperilipin antibody. Calyculin promoted the accumulation of phosphorylated perilipin in a concentration- (B) and time- (D) dependent manner. The blots shown are representatives of 3 separate experiments; p-peri A, phosphorylated perilipin A; peri A, perilipin A; peri B, perilipin B.

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concentration (EC50) of calyculin on lipolytic stimulation was calculated to be 17.4 nM. Further, an increase in lipolysis was undetectable 5 min after the administration of calyculin, whereas an accumulation of phosphorylated perilipin A was obvious at the same time (Fig. 1D). In parallel to the lipolytic elevation by 1.7-fold at 15 min, 4.8-fold at 30 min, and 7.4-fold at 60 min after the addition of calyculin (Fig. 1C), the accumulation of phosphorylated perilipin A was time-dependently promoted (Fig. 1D). Thus, the increased lipolytic action of calyculin was accompanied by the promoted phosphorylation of perilipin A in a concentration- and time-responsive manner. In contrast, the accumulation of phosphorylated perilipin B was minimal and thus unlikely responsible for the large and acute increase of lipolysis. 3.2. Inhibition of PPase promotes phosphorylation of perilipins Perilipin A is a major PKA-phosphorylated protein [9] that functions as a triglyceride-substrate factor to facilitate lipolysis in adipocytes on PKA activation [5,15–17]. We examined whether inhibition of the protein dephosphorylation process with PPase inhibitors resulted in an accumulation of phosphorylated perilipin A. Incubation with 20 nM calyculin or 1 μM isoproterenol did not alter the expression level of perilipin A but caused a similar electrophoretic shift of perilipin A from 65 to ~67 kDa (Fig. 2A). Such a change in migration in the low bis concentration gel is characteristic of increased phosphate incorporation in perilipin A [9,32]. 32P loading of adipocytes and autoradiography of SDS-PAGE gels showed that either inhibition of PPase with calyculin or activation of PKA with isoproterenol promoted the incorporation of 32Pi into perilipin A (Fig. 2B), thus directly confirming that perilipin A was hyperphosphorylated. A 1-h incubation with okadaic acid at 0.5 or 1 μM and calyculin at 20 nM induced a considerable phosphorylation of perilipin A, as evidenced by a shift of the protein band from 65 to ~67 kDa (Fig. 2C). Both immunoblotting and autoradiography confirmed that a weakly phosphorylated perilipin A was already detectable in untreated fat cells (Fig. 2A–C). These data suggest that the perilipins are basally phosphorylated but can be induced to readily hyperphosphorylate when phosphatases are inhibited by the PPase inhibitors. In addition to the phosphorylated perilipin A, perilipin B migrated in the gel from 46 to ~47 kDa (Fig. 2C), a phenomena never observed previously, which suggests that perilipin B was also minimally phosphorylated in adipocytes treated with okadaic acid or calyculin. In parallel to promoted phosphorylation of perilipin A, adipocyte lipolysis was elevated by 9.5- or 13.8-fold after incubation for 1 h with 0.5 or 1 μM okadaic acid, an inhibitor specific for PP2A; similarly, the inhibition of PP1/PP2A activities with 20 nM calyculin led to an 11.5-fold increase in lipolysis (Fig. 2D). 3.3. Abrogation of perilipin phosphorylation abolishes lipolysis The alkylation of heterotrimeric PP2A complex with Nethylmaleimide causes a release of the PP2A catalytic subunit and thus elevates the phosphatase activity [33]. By acting

Fig. 2. Perilipin is hyperphosphorylated upon lipolytic stimulation with PPase inhibitors. (A) the experiment was performed in parallel to that for panel B, in the absence of 32P-loading. The migration pattern of phosphorylated perilipin A (67 kDa) upon the addition of calyculin was consistent with that induced by isoproterenol. (B) autoradiograph of phosphorylated perilipin in the fat cake extracted from 32P-loaded adipocytes. Adipocytes were loaded with 100 μCi/ml [32P]orthophosphate for 90 min and incubated for an additional 30 min in the absence (−) or presence (+) of 20 nM calyculin. Stimulation of adipocytes for 5 min with 1 μM isoproterenol served as a positive control to show 32P-labelled perilipin A (67 kDa). The proteins were extracted from the fat cake of 32Plabeled adipocytes, separated on SDS-PAGE, and visualized by autoradiography. (C) immunoblotting of perilipin. After incubation for 1 h, proteins were extracted from the fat cake fraction and subjected to immunoblotting with primary antibody against rat perilipin. The shift from the 65-kDa to the ~67-kDa perilipin A indicates that the hyperphosphorylated form of perilipin (67 kDa) accumulates upon PPase inhibition with okadaic acid or calyculin. Phosphorylation of perilipin B (from 46 to ~47 kDa) appeared weakly. (D) lipolysis assay. 50 μl of packed adipocytes suspended in 500 μl fDMEM were incubated for 1 h with okadaic acid (OA) or calyculin (Caly). Glycerol release in the culture media was assayed as an index of lipolysis and expressed as micromolecules glycerol (means ± S.E., n = 4) per milliliter of packed cell volume (PCV). The blots are representative of 3 separate experiments; Iso, isoproterenol; Caly, calyculin; OA, okadaic acid; peri, perilipin A; *P < 0.001 versus the vehicle control.

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through this machinery, this sulfhydryl modifying reagent attenuates the phosphorylations of numerous phosphoproteins [34,35]. We demonstrated that pre-incubation of adipocytes with 0.5 mM N-ethylmaleimide greatly abrogated the hyperphosphorylation of perilipin (Fig. 3A) and abolished the release of glycerol (Fig. 3B) from adipocytes treated with calyculin. These data highlight that the phosphorylation of perilipin A plays an important role in the lipolytic pathway upon inhibition of PPase with calyculin. 3.4. Simultaneous inhibition of the phosphatase enhances PKA-activated perilipin hyperphosphorylation and lipolysis We next examined whether PPase is involved in the regulation of PKA-mediated lipolysis. When the phosphatase was inhibited by calyculin, simultaneous PKA activation with isoproterenol converted most of native perilipin A (65 kDa) to the hyperphosphorylated species (67 kDa). By contrast, isoproterenol alone caused a relatively lower magnitude of phosphorylated perilipin A (Fig. 4A). In parallel to this great hyperphosphorylation of perilipin A, enhanced lipolysis was stimulated upon co-incubation with calyculin and isoproterenol, as compared to that induced by either isoproterenol or calyculin alone (Fig. 4B). Therefore, upon isoproterenol stimulation, an

Fig. 4. Inhibition of phosphatase further enhances PKA-activated hyperphosphorylation of perilipin A and lipolysis. Rat adipocytes were incubated for 1 h with 20 nM calyculin or 100 nM isoproterenol, or both. (A) proteins were extracted from the fat cake of adipocytes, separated by SDS-PAGE, and immunoblotted with anti-perilipin antibody. In the presence of calyculin to inhibit phosphatase activity, PKA-activation by isoproterenol converted most native perilipin A (65 kDa) to the hyperphosphorylated species (67 kDa). (B) lipolysis assay of glycerol in the media performed in triplicate. Data are the means ± S.E. of 3 separate experiments; Iso, isoproterenol; Caly, calyculin; *P < 0.001, Caly + Iso versus Caly or Iso alone.

endogenous phosphatase behind elicited PKA may participate in the regulation of the steady state of perilipin phosphorylation and, hence, lipolysis response. 3.5. Calyculin stimulates lipolysis without inducing translocation of HSL

Fig. 3. Abrogation of perilipin phosphorylation abolishes lipolysis. Adipocytes were pre-incubated with 0.5 mM N-ethylmaleimide (NEM) and treated for 1 h with 20 nM calyculin (Caly), 0.5 mM NEM, or both. (A) immunoblot of perilipin proteins extracted from the fat cake fraction. Arrow indicates the band shift of phosphorylated perilipin A. (B) lipolysis assay of glycerol in the culture media in triplicate. The data are representative of 3 experiments.

PKA activation causes the translocation of HSL from the cytosol to the lipid droplet [3–5], which enhances lipolysis stimulated by PKA [5]. We sought to examine the subcellular redistribution of HSL after the addition of calyculin or isoproterenol (as a positive control) immunostaining of differentiated rat adipocytes with anti-HSL antibody showed that stimulation with isoproterenol significantly increased immunofluorescent signals (bright ring-loops) of HSL surrounding the lipid droplet peripheries; in contrast, stimulation with calyculin for 5, 15, 20, and 30 min failed to induce HSL translocation (Fig. 5A). Long-period stimulation with isoproterenol or calyculin could distort adipocytes, thus was not adopted. These immunostaining results were further confirmed by immunoblotting of HSL in the cytosolic and fat cake fractions of primary adipocytes (Fig. 5B). Incubation with isoproterenol decreased cytosolic HSL but elevated HSL in the fat cake fractions, which indicates that the translocation of HSL to the lipid droplets occurred on

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Fig. 5. Calyculin fails to induce a subcellular redistribution of HSL in rat differentiated and primary adipocytes. (A) immunofluorescent staining of HSL in differentiated rat adipocytes stimulated with 10 μM isoproterenol for 5 min or with 20 nM calyculin for indicated period. HSL was assembled as bright ringloops surrounding the surface of the lipid droplets only in isoproterenolstimulated cells. Stimulation with 20 nM calyculin for 5, 15, 20, and 30 min did not cause a redistribution of HSL from the cytosol to the lipid droplets. (B) immunoblot of HSL in the subcellular fractions of cytosol (cyto) and fat cake (fat) isolated from primary rat adipocytes treated for 1 h with 1 μM isoproterenol or 20 nM calyculin. Incubation with isoproterenol induced considerable translocation of HSL from the cytosol to fat cake fractions, whereas calyculin failed to cause subcellular redistribution of HSL; Ctrl, vehicle control; Iso, isoproterenol; Caly, calyculin. The data shown are the representatives of 3 experiments.

PKA activation with isoproterenol. By contrast, calyculin failed to cause a redistribution of HSL from the cytosol to the fat cake fraction of primary adipocytes (Fig. 5B). These data collectively confirm that the lipolytic action upon inhibition of PPase with calyculin is not attributable to an accelerated translocation of HSL to the lipid droplets. 4. Discussion Perilipin A coats the lipid droplets in adipocytes and assumes to deny lipases access to the core triglycerides; the phosphorylation of perilipin A by PKA could lead to modification of the lipid droplet surface and thereby allow lipase access to its substrate, thus facilitating lipolysis [1,2]. Perilipin phosphorylation can be regulated by protein kinase [16,32,36] or phosphatase [20,32]. A low level of phosphorylations on serine/

threonine residues of perilipin mediated by PKA [1] or extracellular signal-related kinase [36] may occur in fat cells and is enough in the presence of phosphatase inhibitors to allow phosphorylated perilipin to accumulate. We show that the inhibition of PPase and, hence, the PPase-mediated process of protein dephosphorylation promotes the incorporation of 32Pi into perilipins in adipocytes, confirming that perilipin is hyperphosphorylated. 32Pi incorporation or the electrophoretic migration pattern of this phosphorylated-perilipin A induced by inhibiting PPase with okadaic acid and calyculin is consistent with that induced by activating PKA with isoproterenol. This observation supports that perilipin is constitutively phosphorylated by the kinase and dephosphorylated by the phosphatase in fat cells. Calyculin inhibits both PP1 and PP2A, but okadaic acid below 1 μM only selectively inhibits PP2A in adipocytes [29] and nonadipose cells [30,31]. Thus, the induction of perilipin phosphorylation by calyculin at 5–40 nM or okadaic acid at 0.5–1 μM implies that the PP2A, perhaps in combination with PP1, coordinately accounts for the activity of perilipin phosphatase in intact adipocytes. However, the definitive perilipin phosphatase remains to be further identified. The PPase could function to modulate lipolysis at a proper level under basal conditions by acting to maintain constitutive state of perilipin phosphorylation. The inhibition of PPase with okadaic acid and calyculin results in the phosphorylation of perilipin, which is accompanied by a gradual increase of lipolysis in a concentration- and time-dependent manner and occurs likely before lipolytic activation. The pre-incubation of adipocytes with N-ethylmaleimide greatly abrogates perilipin phosphorylation and thereby abolishes the lipolysis response to calyculin. N-ethylmaleimide modifies the sulfhydryl residue of cysteine and thus may alter the phosphorylation status of the targeting protein, that is, increase the phosphorylation of insulin receptor [37] or Syk protein [38]. The alkylation of heterotrimeric PP2A complex with N-ethylmaleimide results in the release of the PP2A catalytic subunit, thus elevating the phosphatase activity [33]. By eliciting the PP2A and PP2A-mediated dephosphorylation process, this agent can attenuate the phosphorylation of numerous phosphoproteins such as protein kinase A and B [34,35]. Therefore, N-ethylmaleimide-elevated phosphatase activity may account for the abrogation of perilipin phosphorylation and thence the abolishment of the lipolysis response to calyculin. In addition, N-ethylmaleimide inhibits perilipin phosphorylation and lipolysis in adipocytes stimulated by isoproterenol or by isoproterenol plus calyculin (He, J., and Xu, G., unpublished data). This result highlights that the phosphorylation of perilipin A is critical for the lipolytic action upon inhibition of PPase with calyculin. In contrast, perilipin B, a less abundant and shorter variant, is phosphorylated only minimally and thus unlikely accounts for this large increase in lipolysis. However, N-ethylmaleimide has wide effects including that it inhibits several classes of ATPases [39] and of protein transport processes requiring vesicular fusion [40]; it also suppresses PKA-stimulated translocation of HSL to lipid droplets in 3T3L1 adipocytes [4]. Therefore, the ability of N-ethylmaleimide to prevent calyculin induced lipolysis might be mediated through other actions in addition to changes in perilipin phosphorylation.

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The translocation of HSL to the lipid droplets in adipocytes is an important step for PKA-regulated lipolysis [3–5]. Though an earlier report suggested that okadaic acid at high concentration at 10 μM may slightly induce HSL translocation [19], we failed to detect HSL translocation in both differentiated and primary adipocytes treated with calyculin. Instead, we detected a considerable translocation of HSL in these cells on PKA activation with isoproterenol. Therefore, in contrast to PKA activation, inhibition of PPase with calyculin provides a model adipocyte system, which allows dissociate HSL translocation action from lipolysis and more precisely define the role of phosphorylation of perilipin A in lipolysis. HSL translocation is a consequence of PKA activation [3–5] and requires subsequent phosphorylation of HSL at least at the Ser-659 and -660 residues [41]. HSL is phosphorylated by PKA at Ser-563, -659 and -660 residues [42] and is constitutively phosphorylated at Ser-565, the basal site, in the absence of PKA stimulation [43]. Two major studies have demonstrated a much higher activity of PP2A and PP2C than PP1 toward the PKA sites and the basal site (Ser-565) of rat HSL [44,45]. Therefore, calyculin, a more selective PP1 inhibitor [30,31], likely could not predominately inhibit PP2A and PP2C in adipocytes to ensure sufficient accumulation of phosphorylated HSL at the concerted sites, and thereby translocation of HSL fails to occur. Previously, we revealed that fully phosphorylatable perilipin is essential for HSL translocation on PKA activation [5]. The present data additionally suggest that the hyperphosphorylation of perilipin A alone is not sufficient for HSL translocation, which likely requires the simultaneous PKA phosphorylation of both HSL [41] and perilipin A [5]. However, a mutagenesis strategy of HSL or perilipin is methodologically not suitable for primary adipocytes, where lipolysis primarily occurs. Further investigation should be required to elucidate the phosphorylation sites of these proteins for optimal lipolysis but lack of HSL translocation in response to calyculin alternatively in reconstituted nonadipose cells. HSL is not the only lipase for triglyceride hydrolysis. Adipocytes derived from HSL-null mice retain ~ 50% of lipase activity [6]. ATGL is newly identified as an adipose triglyceride lipase that is abundant on the cytosol and lipid droplets in adipocytes [7,8]. Adipose tissue of combined deficiency of HSL and ATGL still retains ~10% lipase activity [7]. Specific activity of HSL for diglycerides is 10 times that for triglycerides [46], whereas ATGL exhibits only weak activity against diglycerides but is predominantly responsible for the initial step of triglyceride hydrolysis [7]. ATGL can be phosphorylated, but in contrast to HSL, this modification is not mediated by PKA [7]. We did not examine the effects of calyculin on the phosphorylations and activities of ATGL and HSL. Based on the results of that calyculin did not induce HSL translocation but stimulated the release of glycerol, a cautious speculation is that, upon calyculin stimulation, ATGL could be activated and hydrolyze triglycerides to generate diglycerides that are then hydrolyzed by HSL; HSL might manipulate the subsequent hydrolysis of diglycerides in the cytoplasm rather than (translocation) at the surface of the lipid droplets. In fact, there is ~40% of the total HSL [25] and abundant ATGL [7] already associated with lipid

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droplets in PKA-unstimulated adipocytes; these lipases, perhaps combined with other unidentified lipases, seem adequate to manipulate lipolysis when perilipin A is phosphorylated upon inhibition of the PPase with calyculin. On the other hand, even though these lipases are already in place and active, they cannot efficiently manipulate lipolysis if perilipin A is not properly phosphorylated. Thus, the previously accepted rate-limiting action of lipase in triglyceride hydrolysis may likely reflect the barrier function of perilipin, which can be reversed upon the phosphorylation of perilipin A. Protein phosphorylation is modulated by kinase and phosphatase. An elevated cAMP [34,47–50] or stimulation with isoproterenol [49,50] not only activates PKA to phosphorylate targeting proteins but also elicits phosphatase to enhance dephosphorylation of numerous phosphoproteins [34,47–50]. Thus, it is rational to speculate that isoproterenol could elicit a phosphatase to preserve the barrier function of perilipin, thereby manipulating a self-restricted lipolysis on PKA activation with isoproterenol. Here, we provide evidence that when the phosphatase is inhibited by calyculin, simultaneous PKA activation with isoproterenol converts most of the perilipin A to the hyperphosphorylated species, which is accompanied by enhanced lipolysis. Apparently, the PPase participates in the lipolytic regulation in isoproterenol-stimulated adipocytes. While PKA mediates phosphorylation of perilipin and ensures lipolysis, the endogenous phosphatase acts contrarily to dephosphorylate perilipin and restrict lipolysis. Therefore, these data suggest that the lipase-catalyzed lipolysis response to hormonal stimulation can be dually regulated by a kinase and a phosphatase, through a strategy to modulate the phosphorylation state of perilipin A at the surface of the lipid droplets in adipocytes. Acknowledgements We thank Dr. Constantine Londos at the U.S. National Institute of Diabetes and Digestive and Kidney Diseases for the kind gifts of anti-perilipin and anti-HSL antibodies. We thank Dr. Londos and Dr. Alan Kimmel for critical discussions and comments on the manuscript. This work was supported by Grants 30270506 and 30370535 from the National Natural Science Foundation of China, and by Grant 5042015 from the Natural Science Foundation of Beijing Province of China. This work was supported by the Program for New Century Excellent Talents in University of the Education Ministry of China (#NECT-04-0023). References [1] C. Londos, D.L. Brasaemle, C.J. Schultz, D.C. Adler-Wailes, D.M. Levin, A.R. Kimmel, C.M. Rondinone, On the control of lipolysis in adipocytes, Ann. N. Y. Acad. Sci. 892 (1999) 155–168. [2] J.T. Tansey, C. Sztalryd, E.M. Hlavin, A.R. Kimmel, C. Londos, The central role of perilipin a in lipid metabolism and adipocyte lipolysis, IUBMB Life 56 (2004) 379–385. [3] J.J. Egan, A.S. Greenberg, M.K. Chang, S.A. Wek, M.C. Moos Jr., C. Londos, Mechanism of hormone-stimulated lipolysis in adipocytes: translocation of hormone-sensitive lipase to the lipid storage droplet, Proc. Natl. Acad. Sci. U. S. A. 89 (1992) 8537–8541.

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