Hormone-sensitive lipase is involved in hepatic cholesteryl ester hydrolysis
2008; Elsevier BV; Volume: 49; Issue: 8 Linguagem: Inglês
10.1194/jlr.m800198-jlr200
ISSN1539-7262
AutoresMotohiro Sekiya, Jun-ichi Osuga, Naoya Yahagi, Hiroaki Okazaki, Yoshiaki Tamura, Masaki Igarashi, Satoru Takase, Kenji Harada, Sachiko Okazaki, Yoko Iizuka, Ken Ohashi, Hiroaki Yagyu, Mitsuyo Okazaki, Takanari Gotoda, Ryozo Nagai, Takashi Kadowaki, Hitoshi Shimano, Nobuhiro Yamada, Shun Ishibashi,
Tópico(s)Adipose Tissue and Metabolism
ResumoHormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lepob/ob/HSL−/−) showed massive accumulation of CE in the liver compared with Lepob/ob/HSL+/+ mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL−/− mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL−/− mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL−/− mice showed reduced activity of CE hydrolase and contained more CE than those from HSL+/+ mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes. Hormone-sensitive lipase (HSL) regulates the hydrolysis of acylglycerol and cholesteryl ester (CE) in various organs, including adipose tissues. However, the hepatic expression level of HSL has been reported to be almost negligible. In the present study, we found that mice lacking both leptin and HSL (Lepob/ob/HSL−/−) showed massive accumulation of CE in the liver compared with Lepob/ob/HSL+/+ mice, while triacylglycerol (TG) accumulation was modest. Similarly, feeding with a high-cholesterol diet induced hepatic CE accumulation in HSL−/− mice. Supporting these observations, we detected significant expression of protein as well as mRNA of HSL in the liver. HSL−/− mice showed reduced activity of CE hydrolase, but not of TG lipase, in the liver compared with wild-type mice. Furthermore, we confirmed the expression of HSL in viable parenchymal cells isolated from wild-type mice. The hepatocytes from HSL−/− mice showed reduced activity of CE hydrolase and contained more CE than those from HSL+/+ mice even without the incubation with lipoproteins. Incubation with LDL further augmented the accumulation of CE in the HSL-deficient hepatocytes. From these results, we conclude that HSL is involved in the hydrolysis of CE in hepatocyes. The mammalian liver plays a central role in maintaining the whole body cholesterol homeostasis. The liver delivers cholesterol to peripheral tissues via VLDL particles, and the excess peripheral cholesterol is transported to the liver through HDL acceptors (1Brown M.S. Kovanen P.T. Goldstein J.L. Regulation of plasma cholesterol by lipoprotein receptors.Science. 1981; 212: 628-635Crossref PubMed Scopus (600) Google Scholar). Cholesterol balance in the liver involves three major processes: 1) the input pathway, which consists of de novo synthesis and lipoprotein uptake from the periphery; 2) the output pathway, which consists of secretion into the circulation as VLDL and into the bile; 3) reversible conversion of cholesterol to cholesteryl esters (CEs). Esterification of cholesterol with long-chain fatty acyl-CoA is accomplished by ACAT (2Chang T.Y. Chang C.C. Cheng D. Acyl-coenzyme A:cholesterol acyltransferase.Annu. Rev. Biochem. 1997; 66: 613-638Crossref PubMed Scopus (439) Google Scholar). Newly formed CEs are secreted as a component of VLDL or are stored in intracellular lipid droplets in the cytoplasm, where CEs undergo a constant cycle of hydrolysis and resynthesis.To date, at least four major CE hydrolases have been identified in the liver and shown to differ in their subcellular localizations, functions, and enzymatic properties. 1) The lysosomal acid lipase (LAL) is involved in the hydrolysis of CEs and triacylglycerols (TGs) delivered to the hepatocytes via receptor-mediated endocytosis of lipoproteins (3Du H. Grabowski G.A. Lysosomal acid lipase and atherosclerosis.Curr. Opin. Lipidol. 2004; 15: 539-544Crossref PubMed Scopus (32) Google Scholar). 2) The cytosolic CE hydrolase from rat liver has also been purified and characterized. This lipase has been considered to be a key enzyme for releasing free cholesterol from the stores of CEs in the cytoplasm (4Ghosh S. Mallonee D.H. Hylemon P.B. Grogan W.M. Molecular cloning and expression of rat hepatic neutral cholesteryl ester hydrolase.Biochim. Biophys. Acta. 1995; 1259: 305-312Crossref PubMed Scopus (56) Google Scholar). 3) The microsomal CE hydrolase has also been purified from rat liver and characterized (5Cristobal S. Ochoa B. Fresnedo O. Purification and properties of a cholesteryl ester hydrolase from rat liver microsomes.J. Lipid Res. 1999; 40: 715-725Abstract Full Text Full Text PDF PubMed Google Scholar). 4) Carboxyl ester lipase (CEL) is another cholesterol esterase present in the hepatic extralysosomal membrane compartment and possesses the capacity to hydrolyze HDL-CE (6Camarota L.M. Chapman J.M. Hui D.Y. Howles P.N. Carboxyl ester lipase cofractionates with scavenger receptor BI in hepatocyte lipid rafts and enhances selective uptake and hydrolysis of cholesteryl esters from HDL3.J. Biol. Chem. 2004; 279: 27599-27606Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar).Hormone-sensitive lipase (HSL) is an intracellular neutral lipase that catalyzes the hydrolysis of cellular CEs as well as TG, diacylglycerol (DG), monoacylglycerols, and other lipids. HSL is diffusely distributed throughout the cytosol, and upon activation, translocation of HSL from the cytosol to lipid droplets occurs (7Holm C. Osterlund T. Laurell H. Contreras J.A. Molecular mechanisms regulating hormone-sensitive lipase and lipolysis.Annu. Rev. Nutr. 2000; 20: 365-393Crossref PubMed Scopus (343) Google Scholar, 8Kraemer F.B. Shen W.J. Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis.J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar). Although HSL was initially identified as an adipose-specific lipase, it has been clarified that HSL is also expressed and functions in a wide variety of organs and cells, including heart, skeletal muscle, adrenal glands, testes, ovaries, pancreatic β-cells, and intestines (8Kraemer F.B. Shen W.J. Hormone-sensitive lipase: control of intracellular tri-(di-)acylglycerol and cholesteryl ester hydrolysis.J. Lipid Res. 2002; 43: 1585-1594Abstract Full Text Full Text PDF PubMed Scopus (371) Google Scholar, 9Grober J. Lucas S. Sorhede-Winzell M. Zaghini I. Mairal A. Contreras J.A. Besnard P. Holm C. Langin D. Hormone-sensitive lipase is a cholesterol esterase of the intestinal mucosa.J. Biol. Chem. 2003; 278: 6510-6515Abstract Full Text Full Text PDF PubMed Scopus (52) Google Scholar, 10Yeaman S.J. Hormone-sensitive lipase—new roles for an old enzyme.Biochem. J. 2004; 379: 11-22Crossref PubMed Scopus (194) Google Scholar). Despite the broad tissue distribution, the liver has been considered to lack HSL (11Holm C. Belfrage P. Fredrikson G. Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue.Biochem. Biophys. Res. Commun. 1987; 148: 99-105Crossref PubMed Scopus (132) Google Scholar), although observations suggesting the importance of HSL in liver were reported (12Mulder H. Sorhede-Winzell M. Contreras J.A. Fex M. Strom K. Ploug T. Galbo H. Arner P. Lundberg C. Sundler F. et al.Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact.J. Biol. Chem. 2003; 278: 36380-36388Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar).Several laboratories, including ours, have generated HSL-deficient (HSL−/−) mice by targeted gene disruption (12Mulder H. Sorhede-Winzell M. Contreras J.A. Fex M. Strom K. Ploug T. Galbo H. Arner P. Lundberg C. Sundler F. et al.Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact.J. Biol. Chem. 2003; 278: 36380-36388Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (498) Google Scholar, 14Wang S.P. Laurin N. Himms-Hagen J. Rudnicki M.A. Levy E. Robert M.F. Pan L. Oligny L. Mitchell G.A. The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.Obes. Res. 2001; 9: 119-128Crossref PubMed Scopus (189) Google Scholar, 15Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.J. Biol. Chem. 2002; 277: 4806-4815Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar). Despite the decreased ability of their adipocytes to release FFAs, these mice were not obese. We have further generated leptin/HSL double mutant mice (Lepob/ob/HSL−/−) by cross-breeding leptin-deficient Lepob/ob mice with HSL−/− mice (16Sekiya M. Osuga J. Okazaki H. Yahagi N. Harada K. Shen W.J. Tamura Y. Tomita S. Iizuka Y. Ohashi K. et al.Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep ob/ob mice.J. Biol. Chem. 2004; 279: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) to elucidate the role of HSL, especially in the setting of obesity. Unexpectedly, the Lepob/ob/HSL−/− mice gained less adiposity than Lepob/ob mice, with massive accumulation of preadipocytes in adipose tissue, and HSL deficiency also suppressed the feeding activity of Lepob/ob mice.Here, we show that HSL plays a pivotal role as a lipase in the liver, especially as a CE hydrolase. Massive accumulation of CEs was observed in the liver of Lepob/ob/HSL−/− mice, while the hepatic TG content of Lepob/ob/HSL−/− mice was similar to that of Lepob/ob/HSL+/+ mice. Moreover, feeding diets containing high cholesterol resulted in the accumulation of hepatic CEs in HSL−/− mice. In accordance with these results, CE hydrolase activity was decreased by HSL deficiency, while TG lipase activity was unchanged. These results suggested that HSL could serve as a CE hydrolase in hepatocytes. As expected, the expression of HSL is detected in liver homogenates, and parenchymal cells (PCs) contain more HSL protein than nonparenchymal cells (NPCs). Furthermore, to eliminate indirect effects from other tissues, we isolated primary hepatocytes. The CE hydrolase activity is reduced in the hepatocytes from HSL−/− mice. Intracellular CE content is increased even in the absence of lipoproteins in the hepatocytes from HSL−/− mice, and upon exposure to LDL, a more pronounced increase was observed. Based on these findings, we conclude that HSL is definitely expressed in hepatocytes and involved in CE hydrolysis in the liver.EXPERIMENTAL PROCEDURESAnimalsCross-breeding experiments used leptin-deficient Lepob/ob mice. HSL−/− mice (13Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (498) Google Scholar) and Lepob/ob/HSL−/− mice (16Sekiya M. Osuga J. Okazaki H. Yahagi N. Harada K. Shen W.J. Tamura Y. Tomita S. Iizuka Y. Ohashi K. et al.Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep ob/ob mice.J. Biol. Chem. 2004; 279: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar) were generated, and genotyping was performed as described previously (16Sekiya M. Osuga J. Okazaki H. Yahagi N. Harada K. Shen W.J. Tamura Y. Tomita S. Iizuka Y. Ohashi K. et al.Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep ob/ob mice.J. Biol. Chem. 2004; 279: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). Mice were housed in a temperature-controlled environment with a 12 h light/dark cycle and allowed free access to water and a standard chow diet (Oriental MF, composed of 60% carbohydrate, 13% fat, and 27% protein on a caloric basis; Oriental Yeast, Tokyo, Japan). Mice were euthanized at 16 weeks after a 6 h fast, except in the fasting/refeeding study, in which mice were starved for 24 h or refed for 12 h after a 24 h starvation.Dietary cholesterol load experimentsEight-week-old female HSL−/− mice and their littermate wild-type mice were fed a standard chow with or without 2% cholesterol for 7 weeks. Mice were euthanized after a 12 h starvation. All experiments were performed in accordance with our institutional guidelines.Liver lipid contentLiver lipid was extracted by the chloroform-methanol method, and total cholesterol and free cholesterol were determined by fluorometric microassay according to a modified method of Heider and Boyett (17Heider J.G. Boyett R.L. The picomole determination of free and total cholesterol in cells in culture.J. Lipid Res. 1978; 19: 514-518Abstract Full Text PDF PubMed Google Scholar), with some modifications as described previously (18Yagyu H. Kitamine T. Osuga J. Tozawa R. Chen Z. Kaji Y. Oka T. Perrey S. Tamura Y. Ohashi K. et al.Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.J. Biol. Chem. 2000; 275: 21324-21330Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar).Immunoblotting of HSLLivers (two mice for each genotype) were excised from 16 week old male mice after 6 h of fasting, homogenized in buffer A (20 mM Tris-HCl, pH 7.0, 250 mM sucrose, 5 μg/ml leupeptin, and 2.8 μg/ml aprotinin), and microcentrifuged at 14,000×g for 15 min. Fifty micrograms of the supernatant fraction was subjected to SDS-PAGE analysis. Homogenate (10 μg) from epididymal white adipose tissues of a wild-type male mouse (16 weeks old) was used as a control. Immunoblot analysis was performed using an ECL kit (Amersham Biosciences, Piscataway, NJ) (13Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (498) Google Scholar).Hepatocyte isolationIsolation of viable PCs and NPCs from mouse liver was performed as described previously (19Pretlow T.G. Pretlow T.P. Cell separation by gradient centrifugation methods.Methods Enzymol. 1989; 171: 462-482Crossref PubMed Scopus (12) Google Scholar, 20Matsumoto K. Sano H. Nagai R. Suzuki H. Kodama T. Yoshida M. Ueda S. Smedsrod B. Horiuchi S. Endocytic uptake of advanced glycation end products by mouse liver sinusoidal endothelial cells is mediated by a scavenger receptor distinct from the macrophage scavenger receptor class A.Biochem. J. 2000; 352: 233-240Crossref PubMed Scopus (49) Google Scholar). In brief, the portal vein was cannulated, and the liver was perfused with 0.05% collagenase (Wako Pure Chemicals, Tokyo, Japan). After removing undigested tissues, the cell suspension was centrifuged at 100×g for 2 min. The pellet was used for PC preparation, while for NPC preparation the supernatant was filtered through a nylon mesh to eliminate PCs. The cell suspension was layered on top of 70% (v/v) Percoll (Amersham Bioscience, Tokyo, Japan) and then centrifuged at 800×g for 10 min. The pellet was saved as intact PCs. The supernatant located on the top of Percoll, which contained most of the NPCs but also some PCs and debris, was further centrifuged through a two-step Percoll gradient (25% + 50%) at 800×g for 30 min. A pure NPC band at the interface between the two density cushions was collected. The cells were washed twice with PBS and pelleted by centrifugation. The purity of the cells was morphologically assessed by light microscopic examination. More than 90% of the cells in the PC fraction were hepatocytes, whereas less than 10% of the cells in the NPC fraction were hepatocytes. The cell pellet was redissolved in buffer A and sonicated with a Branson sonifier at power 2 for two 10 s pulses. After microcentrifugation at 14,000×g for 10 min, an aliquot of the supernatant was analyzed by immunoblot analysis.Northern blot analysisTotal RNA was isolated using Trizol Reagent (Life Technologies, Helgerman, CT). Ten microgram RNA samples equally pooled from four to six mice of each genotype were electrophoresed on a 1% agarose gel containing formaldehyde and transferred to a nylon membrane. The membranes were hybridized with probes that were labeled with [α-32P]dCTP using the Megaprime DNA Labeling System kit (Amersham Biosciences) in Rapid-hyb Buffer (Amersham Biosciences) and analyzed using a BAS2000 Bioimaging Analyzer (Fuji Photo Film, Tokyo, Japan). Loading was normalized by the expression of 36B4 (acidic ribosomal phosphoprotein P0) mRNA.TG lipase and neutral CE hydrolase activity assayEnzyme activity was assayed basically as described previously (13Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (498) Google Scholar) with some modifications in the sample preparation. In brief, liver S-100 protein in buffer A was obtained by ultracentrifugation at 100,000×g for 45 min. Buffer exchange to PBS containing 1 mM EDTA was carried out by dialysis for 4 h, because the liver homogenate turned out to include some unknown factor(s) that interfered with assay linearity (data not shown). The samples were incubated at 37°C for 30–60 min in a final volume of 200 μl of a reaction mixture containing 105 μmol/l tri[3H]oleoylglycerol (99.4 μCi/μmol), 23.7 μmol/l lecithin, 12.5 μmol/l sodium taurocholate, 1 mol/l NaCl, and 85 mmol/l potassium phosphate (pH 7.0). The high concentration of NaCl was included to inactivate LPL. CE hydrolase activity was measured using a reaction mixture containing 6.14 μmol/l cholesterol [1-14C]oleate (48.8 μCi/μmol).Microsomal ACAT activityLiver microsomal protein was obtained by ultracentrifugation at 100,000 g for 45 min. The precipitates were resuspended and ultracentrifuged again at 100,000×g for 45 min. The precipitates were resuspended and used for the assay. ACAT activity in microsomes was determined by the rate of incorporation of [1-14C]oleoyl-CoA into the CE fraction according to Yagyu et al. (18Yagyu H. Kitamine T. Osuga J. Tozawa R. Chen Z. Kaji Y. Oka T. Perrey S. Tamura Y. Ohashi K. et al.Absence of ACAT-1 attenuates atherosclerosis but causes dry eye and cutaneous xanthomatosis in mice with congenital hyperlipidemia.J. Biol. Chem. 2000; 275: 21324-21330Abstract Full Text Full Text PDF PubMed Scopus (156) Google Scholar).Biochemical analysesBlood was collected from the retro-orbital venous plexus after a 6 h or a 12 h fast. Cholesterol (Determiner TC; Kyowa Medex, Tokyo, Japan), TG (TG LH; Wako Pure Chemicals), FFA (NEFA C; Wako Pure Chemicals), and alanine aminotransferase (ALT) (transaminase C-II: Pyruvate oxidase Pediococcus sp. (POP) N-(2-hydroxy-3-sulfopropyl)-m-toluidine (TOOS) method; Wako Pure Chemicals) were measured enzymatically. HPLC was performed as described previously (21Magoori K. Kang M.J. Ito M.R. Kakuuchi H. Ioka R.X. Kamataki A. Kim D.H. Asaba H. Iwasaki S. Takei Y.A. et al.Severe hypercholesterolemia, impaired fat tolerance, and advanced atherosclerosis in mice lacking both low density lipoprotein receptor-related protein 5 and apolipoprotein E.J. Biol. Chem. 2003; 278: 11331-11336Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar).TLCLiver lipid was extracted by the chloroform-methanol method, and aliquots were pooled for each genotype. Evaporated lipid was dissolved in chloroform, and lipid from 0.6 mg of liver tissue was loaded in each lane. Lipids were separated by TLC with hexane-diethylether-acetic acid (80:20:1) or chloroform-acetone-acetic acid (96:4:1) as solvent. Visualization was done with 10% sulfuric acid.HistologyMice (16 weeks) were euthanized after a 6 h fast, and their livers were excised and fixed in 10% neutral buffered formalin and embedded in paraffin. Sections were stained with hematoxylin-eosin.Primary culture of hepatocytes and CE formationHepatocytes were isolated from nonfasted HSL−/− mice and their littermate HSL+/+ mice (10 weeks) by the collagenase perfusion method as described previously (22Sekiya M. Yahagi N. Matsuzaka T. Takeuchi Y. Nakagawa Y. Takahashi H. Okazaki H. Iizuka Y. Ohashi K. Gotoda T. et al.SREBP-1-independent regulation of lipogenic gene expression in adipocytes.J. Lipid Res. 2007; 48: 1581-1591Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Cells were resuspended in Williams' E medium supplemented with 5% fetal calf serum, and seeded on collagen-coated dishes at a final density of 2 × 104 cells/cm2, and attached for 6 h.For the neutral CE hydrolase assay, the attached cells were maintained in Medium 199 containing Earle's salts (Invitrogen) and 5% fetal calf serum. After incubation for 20 h, the medium was replaced with the same medium and the cells were further incubated for 12 h. The cells were washed twice with PBS, scraped into buffer A, and sonicated with a Branson sonifier at power 2 for two 10 s pulses. After microcentrifugation at 14,000 g for 10 min, an aliquot of the supernatant was analyzed for neutral CE hydrolase assay.For CE formation, attached cells were incubated in Williams' E medium containing 5% human lipoprotein-deficient serum (LPDS) for 48 h and subsequently incubated in the same medium containing human LDL (100 μg/ml) and [1-14C]oleate-albumin complex with vehicle or 10 μM CS-505, an ACAT inhibitor, which were provided by Sankyo and Kyoto Pharmaceuticals (Tokyo, Japan), for 24 h as described previously (23Perrey S. Ishibashi S. Kitamine T. Osuga J. Yagyu H. Chen Z. Shionoiri F. Iizuka Y. Yahagi N. Tamura Y. et al.The LDL receptor is the major pathway for beta-VLDL uptake by mouse peritoneal macrophages.Atherosclerosis. 2001; 154: 51-60Abstract Full Text Full Text PDF PubMed Scopus (21) Google Scholar) with minor modifications. Intracellular lipids were separated by TLC, and spots corresponding to the CE fraction were scraped and radioactivities were measured. LPDS (d > 1.21 g/ml) and LDL (d = 1.019–1.063 g/ml) were isolated with sequential ultracentrifugation from plasma of healthy human volunteer subjects (24Havel R.J. Eder H.A. Bragdon J.H. The distribution and chemical composition of ultracentrifugally separated lipoproteins in human serum.J. Clin. Invest. 1955; 34: 1345-1353Crossref PubMed Scopus (6460) Google Scholar). Informed consent was obtained from the volunteers according to our institutional guideline for the use of human materials.StatisticsStatistical differences between groups were analyzed by one-way ANOVA and a posthoc Tukey-Kramer test, unless stated otherwise.RESULTSBoth leptin deficiency and dietary cholesterol load induce massive accumulation of CEs in the liver of HSL−/− miceLeptin-deficient Lepob/ob mice exhibit many characteristics of obesity, including hepatic steatosis (25Bray G.A. York D.A. Hypothalamic and genetic obesity in experimental animals: an autonomic and endocrine hypothesis.Physiol. Rev. 1979; 59: 719-809Crossref PubMed Scopus (960) Google Scholar). To determine the effect of HSL deficiency on hepatic lipid metabolism, especially in a leptin-deficient obesity model, HSL−/− mice were cross-bred with leptin-deficient Lepob/ob mice and the liver lipid content of each genotype was measured (6 h fasted mice; Fig. 1A). To our surprise, in the Lepob/ob background, the hepatic CE content was remarkably increased (173% for males and 214% for females) in Lepob/ob/HSL−/− mice compared with Lepob/ob/HSL+/+ mice. There was no difference in acylglycerol content between Lepob/ob/HSL+/+ and Lepob/ob/HSL−/− mice. In the Lep+/+ background, acylglycerol content was reduced in HSL−/− mice compared with HSL−/− mice, consistent with previous reports (26Haemmerle G. Zimmermann R. Strauss J.G. Kratky D. Riederer M. Knipping G. Zechner R. Hormone-sensitive lipase deficiency in mice changes the plasma lipid profile by affecting the tissue-specific expression pattern of lipoprotein lipase in adipose tissue and muscle.J. Biol. Chem. 2002; 277: 12946-12952Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar, 27Voshol P.J. Haemmerle G. Ouwens D.M. Zimmermann R. Zechner R. Teusink B. Maassen J.A. Havekes L.M. Romijn J.A. Increased hepatic insulin sensitivity together with decreased hepatic triglyceride stores in hormone-sensitive lipase-deficient mice.Endocrinology. 2003; 144: 3456-3462Crossref PubMed Scopus (91) Google Scholar). There was a trend toward an increase in hepatic CE content in HSL−/− compared with HSL+/+ mice, especially in female mice (17% for males and 137% for females). Free cholesterol content was not affected by either the absence or the presence of HSL or leptin.HSL−/− mice have reduced plasma FFA levels caused by the impediment of the mobilization of FFA from adipose tissues (12Mulder H. Sorhede-Winzell M. Contreras J.A. Fex M. Strom K. Ploug T. Galbo H. Arner P. Lundberg C. Sundler F. et al.Hormone-sensitive lipase null mice exhibit signs of impaired insulin sensitivity whereas insulin secretion is intact.J. Biol. Chem. 2003; 278: 36380-36388Abstract Full Text Full Text PDF PubMed Scopus (100) Google Scholar, 13Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. Fujimoto A. Shionoiri F. Yahagi N. Kraemer F.B. Tsutsumi O. et al.Targeted disruption of hormone-sensitive lipase results in male sterility and adipocyte hypertrophy, but not in obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 787-792Crossref PubMed Scopus (498) Google Scholar, 14Wang S.P. Laurin N. Himms-Hagen J. Rudnicki M.A. Levy E. Robert M.F. Pan L. Oligny L. Mitchell G.A. The adipose tissue phenotype of hormone-sensitive lipase deficiency in mice.Obes. Res. 2001; 9: 119-128Crossref PubMed Scopus (189) Google Scholar, 15Haemmerle G. Zimmermann R. Hayn M. Theussl C. Waeg G. Wagner E. Sattler W. Magin T.M. Wagner E.F. Zechner R. Hormone-sensitive lipase deficiency in mice causes diglyceride accumulation in adipose tissue, muscle, and testis.J. Biol. Chem. 2002; 277: 4806-4815Abstract Full Text Full Text PDF PubMed Scopus (480) Google Scholar, 16Sekiya M. Osuga J. Okazaki H. Yahagi N. Harada K. Shen W.J. Tamura Y. Tomita S. Iizuka Y. Ohashi K. et al.Absence of hormone-sensitive lipase inhibits obesity and adipogenesis in Lep ob/ob mice.J. Biol. Chem. 2004; 279: 15084-15090Abstract Full Text Full Text PDF PubMed Scopus (48) Google Scholar). In the fasted state, HSL normally stimulates adipose lipolysis, thus increasing the influx of FFA to the liver and contributing to hepatic TG accumulation. Therefore, we evaluated both fasted and refed states (fasting, 24 h of fasting; refeeding, 12 h of feeding after 24 h of fasting; Fig. 1B). In the fasted state, hepatic TG and CE tended to accumulate to a greater extent, and the differences between HSL+/+ and HSL−/− mice were more pronounced than in the refed state. In both states, the accumulation of CEs in the HSL−/− mice described above was conserved.Moreover, hepatic accumulation of CEs in HSL−/− mice was confirmed with the diet-induced model. HSL−/− mice were challenged with dietary cholesterol (2%) for 7 weeks (euthanized after a 12 h fast; Fig. 1C). Dietary cholesterol induced hepatic CE accumulation in HSL−/− mice (217% increase).HSL is expressed in PCsLiver has been reported to express HSL at a negligible level (11Holm C. Belfrage P. Fredrikson G. Immunological evidence for the presence of hormone-sensitive lipase in rat tissues other than adipose tissue.Biochem. Biophys. Res. Commun. 1987; 148: 99-105Crossref PubMed Scopus (132) Google Scholar), whereas our data suggested that there was hepatic HSL expression. Therefore, we performed immunoblot analysis of liver homogenates (Fig. 2A). The hepatic expression of HSL was detected in both Lep+/+ and Lepob/ob mice using our polyclonal anti-HSL antibody, and the expression level was not affected by the presence or absence of leptin. Quantification of the immunoblotting signals indicated that the expression levels of HSL in liver were approximately 5% of those in adipose tissue.Fig. 2Immunoblot analysis of HSL protein in the liver. A: Homogenate of liver (50 μg) was subjected to SDS-PAGE in duplicate for each genotype. In the right lane, homogenate (10 μg) of epididymal white adipose tissue (WAT) from wild-type male mice was loaded as a control. B: Immunoblot analysis of different types of liver cells and murine hepatocyte cell lines using polyclonal anti-HSL antibody. Homogenate (20 μg) of each cell population was loaded. In the right lane, homogenate (4 μg) of epididymal white adipose tissue from wild-type male mice was loaded as a control. PC, parenchymal cell; NPC, nonparenchymal cell; H2.35, H2.35 hepatocyte cell line; McARH, McARH-7777 hepatoma cell line; MPM, mouse peritoneal macrophage.View Large Image Figure ViewerDownload Hi-res image Download (PPT)HSL has been considered to be present in macrophages (13Osuga J. Ishibashi S. Oka T. Yagyu H. Tozawa R. F
Referência(s)