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• W2029718447 abstract "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 β-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. 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 β-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. hormone-sensitive lipase polyacrylamide gel electrophoresis packed cell volume Krebs-Ringer Hepes buffer perilipin NH2-terminal 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.Yeaman S.J. Smith G.M. Jepson C.A. Wood S.L. Emmison N. Adv. Enzyme Regul. 1994; 34: 355-370Crossref PubMed Scopus (65) Google Scholar and 2.Langin D. Holm C. Lafontan M. Proc. Nutr. Soc. 1996; 55: 93-109Crossref PubMed Scopus (142) Google Scholar), the activation of purified HSL by phosphorylation in vitro(3.Fredrikson G. Stralfors P. Nilsson N.O. Belfrage P. J. Biol. Chem. 1981; 256: 6311-6320Abstract Full Text PDF PubMed Google Scholar) is insufficient to account for the stimulation of lipolysisin vivo (4.Nilsson N.O. Stralfors P. Fredrikson G. Belfrage P. FEBS Lett. 1980; 111: 125-130Crossref PubMed Scopus (95) Google Scholar), 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.Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos Jr., M.C. Londos C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8537-8541Crossref PubMed Scopus (343) Google Scholar), 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.Londos C. Brasaemle D.L. Gruia-Gray J. Servetnick D.A. Schultz C.J. Levin D.M. Kimmel A.R. Biochem. Soc. Trans. 1995; 23: 611-615Crossref PubMed Scopus (100) Google Scholar). In addition to the well established mechanism of cAMP-dependent activation of HSL, Okuda et al.(7.Okuda H. Yanagi I. Fujii S. J. Biochem. (Tokyo). 1966; 59: 438-442Crossref PubMed Scopus (21) Google Scholar, 8.Okuda H. Fujii S. J. Biochem. (Tokyo). 1968; 64: 377-385Crossref PubMed Scopus (76) Google Scholar) 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.Wise L.S. Jungas R.L. J. Biol. Chem. 1978; 253: 2624-2627Abstract Full Text PDF PubMed Google Scholar) 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.Barber T. Dwyer N.K. Levin D. Londos C. Blanchette-Mackie E.J. Mol. Biol. Cell. 1995; 6: 437AGoogle Scholar, 11.Fong T.H. Wang S.M. Lin H.S. J. Cell. Biochem. 1996; 63: 366-373Crossref PubMed Scopus (14) Google Scholar). 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.Morimoto C. Tsujita T. Okuda H. J. Lipid Res. 1997; 38: 132-138Abstract Full Text PDF PubMed Google Scholar), 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.Greenberg A.S. Egan J.J. Wek S.A. Garty N.B. Blanchette-Mackie E.J. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar). 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.Servetnick D.A. Brasaemle D.L. Gruia-Gray J. Kimmel A.R. Wolff J. Londos C. J. Biol. Chem. 1995; 270: 16970-16973Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar). 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.Egan J.J. Greenberg A.S. Chang M.K. Londos C. J. Biol. Chem. 1990; 265: 18769-18775Abstract Full Text PDF PubMed Google Scholar, 16.Mooney R.A. Bordwell K.L. Biochem. J. 1991; 274: 433-438Crossref PubMed Scopus (10) Google Scholar), although different phosphatases may be involved (17.Clifford G.M. McCormick D.K.T. Londos C. Vernon R.J. Yeaman S.J. FEBS Lett. 1998; 435: 125-129Crossref PubMed Scopus (29) Google Scholar). Recently, tumor necrosis factor α 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.Souza S.C. Yamamoto M.T. Franciosa M.D. Lien P. Greenberg A.S. Diabetes. 1998; 47: 691-695Crossref PubMed Scopus (151) Google Scholar). Furthermore overexpression of perilipins A and B in 3T3-L1 adipocytes blocked the ability of tumor necrosis factor α, but not isoproterenol (a β-adrenergic agonist), to increase lipolysis (19.Souza S.C. Moitoso de Vargas L. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). 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.Londos C. Brasaemle D.L. Gruia-Gray J. Servetnick D.A. Schultz C.J. Levin D.M. Kimmel A.R. Biochem. Soc. Trans. 1995; 23: 611-615Crossref PubMed Scopus (100) Google Scholar, 18.Souza S.C. Yamamoto M.T. Franciosa M.D. Lien P. Greenberg A.S. Diabetes. 1998; 47: 691-695Crossref PubMed Scopus (151) Google Scholar). 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. [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.Servetnick D.A. Brasaemle D.L. Gruia-Gray J. Kimmel A.R. Wolff J. Londos C. J. Biol. Chem. 1995; 270: 16970-16973Abstract Full Text Full Text PDF PubMed Scopus (161) Google Scholar), as was an antibody raised against rat HSL/bacterial fusion protein (20.Kraemer F.B. Patel S. Saedi M.S. Sztalryd C. J. Lipid Res. 1993; 34: 663-671Abstract Full Text PDF PubMed Google Scholar). 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). Adipocytes were isolated by collagenase digestion of the epididymal fat pads (21.Rodbell M. J. Biol. Chem. 1964; 239: 375-380Abstract Full Text PDF PubMed Google Scholar) 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 mmMgCl2, 3% bovine serum albumin, and 2 mmglucose (reduced phosphate KRH). 200 nm adenosine was included to suppress cAMP production and stimulation of cAMP-dependent protein kinase activity (22.Honnor R.C. Dhillon G.S. Londos C. J. Biol. Chem. 1985; 260: 15130-15138Abstract Full Text PDF PubMed Google Scholar). 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. 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 mmTris-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. Adipocytes (200 μl PCV/ml) were loaded with [32P]Pi, 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 was performed using a Tris/glycine buffer system with Hoeffer mini-gel apparatus (23.Laemmli U.K. Favre M. J. Mol. Biol. 1973; 80: 575-599Crossref PubMed Scopus (3010) Google Scholar). 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. 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.McGowan M.W. Artiss J.D. Strandbergh D.R. Zak B. Clin. Chem. 1983; 29: 538-542Crossref PubMed Scopus (1062) Google Scholar. Glycerol release is expressed as nanomoles glycerol ml−1 PCV min−1. 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.1 A). 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. 1 B). 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. 1 B). 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.Smith G.M. Garton A.J. Aitken A. Yeaman S.J. FEBS Lett. 1996; 396: 90-94Crossref PubMed Scopus (27) Google Scholar), 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. 2G. M. Clifford and S. J. Yeaman, unpublished results. Figure 2Time 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 μmisoproterenol. 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.View Large Image Figure ViewerDownload Hi-res image Download (PPT) 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.3 B). 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). Consistent with previous reports (13.Greenberg A.S. Egan J.J. Wek S.A. Garty N.B. Blanchette-Mackie E.J. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar, 15.Egan J.J. Greenberg A.S. Chang M.K. Londos C. J. Biol. Chem. 1990; 265: 18769-18775Abstract Full Text PDF PubMed Google Scholar), 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.Greenberg A.S. Egan J.J. Wek S.A. Garty N.B. Blanchette-Mackie E.J. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar). 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.5 A) 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.5 B). 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. 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. 5 C), 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. 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 μmisoproterenol 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. 3 A). Perilipin was undergoing multiple phosphorylation upon lipolytic stimulation in these cells as shown by the decrease in electrophoretic mobility (Fig.5 C). 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. 3 B). 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. This study demonstrates that the translocation of HSL in response to the β-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.Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos Jr., M.C. Londos C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8537-8541Crossref PubMed Scopus (343) Google Scholar). 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.Egan J.J. Greenberg A.S. Chang M.K. Londos C. J. Biol. Chem. 1990; 265: 18769-18775Abstract Full Text PDF PubMed Google Scholar, 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.Egan J.J. Greenberg A.S. Chang M.K. Wek S.A. Moos Jr., M.C. Londos C. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 8537-8541Crossref PubMed Scopus (343) Google Scholar), 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.Shen W.J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Crossref PubMed Scopus (176) Google Scholar). 3C. J. Schultz and C. Londos, unpublished results. 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.Souza S.C. Moitoso de Vargas L. Yamamoto M.T. Lien P. Franciosa M.D. Moss L.G. Greenberg A.S. J. Biol. Chem. 1998; 273: 24665-24669Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar, 27.Londos C. Basaemle D.L. Schultz C.J. Segrest J.P. Kimmel A.R. Semin. Cell Dev. Biol. 1999; 10: 51-58Crossref PubMed Scopus (364) Google Scholar). 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.Greenberg A.S. Egan J.J. Wek S.A. Garty N.B. Blanchette-Mackie E.J. Londos C. J. Biol. Chem. 1991; 266: 11341-11346Abstract Full Text PDF PubMed Google Scholar). 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.Barber T. Dwyer N.K. Levin D. Londos C. Blanchette-Mackie E.J. Mol. Biol. Cell. 1995; 6: 437AGoogle Scholar) and Leydig cells (28.Wang S.M. Fong T.H. Hsu S.Y. Chien C.L. Wu J.C. J. Cell. Biochem. 1997; 67: 84-91Crossref PubMed Scopus (17) Google Scholar), 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.Giudicelli Y. Pecquery R. Eur. J. Biochem. 1978; 90: 413-419Crossref PubMed Scopus (78) Google Scholar, 30.Gonzalez J. DeMartinis F.D. Exp. Ageing Res. 1978; 4: 455-477Crossref PubMed Scopus (11) Google Scholar). 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.Gruen R. Kava R. Greenwood M.R.C. Metabolism. 1980; 29: 246-253Abstract Full Text PDF PubMed Scopus (13) Google Scholar). 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.Dax E.M. Partilla J.S. Gregerman R.I. J. Lipid Res. 1981; 22: 934-943Abstract Full Text PDF PubMed Google Scholar). 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.Blanchette-Mackie E.J. Dwyer N.K. Barber T. Coxey R.A. Takeda T. Rondinone C.M. Theodorakis J.L. Greenberg A.S. Londos C. J. Lipid. Res. 1995; 36: 1211-1226Abstract Full Text PDF PubMed Google Scholar). Similarly, as rats mature there may be changes in the levels of proteins which interact with HSL such as adipocyte lipid-binding protein (26.Shen W.J. Sridhar K. Bernlohr D.A. Kraemer F.B. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 5528-5532Crossref PubMed Scopus (176) Google Scholar), or lipotransin, which may localize HSL to the lipid droplet in a hormone-sensitive manner (34.Syu L.-J. Saltiel A.R. Mol. Cell. 1999; 4: 109-115Abstract Full Text Full Text PDF PubMed Scopus (58) Google Scholar)." @default.
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• W2029718447 date "2000-02-01" @default.
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• W2029718447 title "Translocation of Hormone-sensitive Lipase and Perilipin upon Lipolytic Stimulation of Rat Adipocytes" @default.
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• W2029718447 doi "https://doi.org/10.1074/jbc.275.7.5011" @default.
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