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• W2084516396 abstract "Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart.Background: Perilipin family proteins are important in determining the properties of lipid droplets (LDs).Results: Perilipin 5-deficient mice lack detectable LDs, exhibit enhanced fatty acid oxidation, and suffer increased ROS production in the heart.Conclusion: Perilipin 5 protects the heart from oxidative burden by sequestering fatty acid from excessive oxidation.Significance: These findings may help to increase understanding of the functions of non-adipose LDs. Lipid droplets (LDs) are ubiquitous organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. The properties of LDs vary greatly among tissues, and LD-binding proteins, the perilipin family in particular, play critical roles in determining such diversity. Overaccumulation of TAG in LDs of non-adipose tissues may cause lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. However, the physiological significance of non-adipose LDs in a normal state is poorly understood. To address this issue, we generated and characterized mice deficient in perilipin 5 (Plin5), a member of the perilipin family particularly abundant in the heart. The mutant mice lacked detectable LDs, containing significantly less TAG in the heart. Particulate structures containing another LD-binding protein, Plin2, but negative for lipid staining, remained in mutant mice hearts. LDs were recovered by perfusing the heart with an inhibitor of lipase. Cultured cardiomyocytes from Plin5-null mice more actively oxidized fatty acid than those of wild-type mice. Production of reactive oxygen species was increased in the mutant mice hearts, leading to a greater decline in heart function with age. This was, however, reduced by the administration of N-acetylcysteine, a precursor of an antioxidant, glutathione. Thus, we conclude that Plin5 is essential for maintaining LDs at detectable sizes in the heart, by antagonizing lipase(s). LDs in turn prevent excess reactive oxygen species production by sequestering fatty acid from oxidation and hence suppress oxidative burden to the heart. Background: Perilipin family proteins are important in determining the properties of lipid droplets (LDs). Results: Perilipin 5-deficient mice lack detectable LDs, exhibit enhanced fatty acid oxidation, and suffer increased ROS production in the heart. Conclusion: Perilipin 5 protects the heart from oxidative burden by sequestering fatty acid from excessive oxidation. Significance: These findings may help to increase understanding of the functions of non-adipose LDs. Lipid droplets (LDs) 4The abbreviations used are: LDlipid dropletTAGtriacylglycerolWATwhite adipose tissueFAfatty acidBATbrown adipose tissueATGLadipose triacylglycerol lipaseHSLhormone-sensitive lipaseROSreactive oxygen speciesNACN-acetylcysteineOROOil Red OBELbromoenol lactoneTBARSthiobarbituric acid-reactive substanceLVID;dleft ventricular end-diastolic dimensionLVID;sleft ventricular end-systolic dimensionFSleft ventricular fractional shorteningASMacid-soluble material. are cellular organelles storing neutral lipids, including triacylglycerol (TAG) and cholesterol ester. LDs are found in nearly all cell types, but their properties vary greatly among tissues. White adipose tissue (WAT) has large unilocular LDs that store an enormous amount of TAG in case of increased energy demand. LDs of other tissues, however, are usually much smaller. Their physiological significance is less well understood, although a possible role is in sequestering fatty acid (FA) in a chemically inert form, TAG, to circumvent the lipotoxicity of FA and its derivatives (1Schaffer J.E. Lipotoxicity. When tissues overeat.Curr. Opin. Lipidol. 2003; 14: 281-287Crossref PubMed Scopus (706) Google Scholar). However, excess accumulation of TAG, and hence aberrant development of LDs, often causes lipotoxicity, leading to diseases such as diabetes and cardiomyopathy. lipid droplet triacylglycerol white adipose tissue fatty acid brown adipose tissue adipose triacylglycerol lipase hormone-sensitive lipase reactive oxygen species N-acetylcysteine Oil Red O bromoenol lactone thiobarbituric acid-reactive substance left ventricular end-diastolic dimension left ventricular end-systolic dimension left ventricular fractional shortening acid-soluble material. LDs carry a defined set of surface-binding proteins whose compositions differ among cell types. The perilipin family, conventionally called the PAT family, is a representative group of LD-binding proteins composed of five members (2Brasaemle D.L. Thematic review series. Adipocyte biology. The perilipin family of structural lipid droplet proteins. Stabilization of lipid droplets and control of lipolysis.J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). The recently proposed unified nomenclature (3Kimmel A.R. Brasaemle D.L. McAndrews-Hill M. Sztalryd C. Londos C. Adoption of perilipin as a unifying nomenclature for the mammalian PAT family of intracellular lipid storage droplet proteins.J. Lipid Res. 2010; 51: 468-471Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar) names them PLIN1 (the classic perilipin), PLIN2 (also named ADRP, ADFP, or adipophilin), PLIN3 (Tip47, PP17, or M6PRBP), PLIN4 (S3–12), and PLIN5 (MLDP, OXPAT, LSDP5, or PAT1). These proteins have related amino acid sequences, particularly in the amino-terminal region called the PAT1 domain (2Brasaemle D.L. Thematic review series. Adipocyte biology. The perilipin family of structural lipid droplet proteins. Stabilization of lipid droplets and control of lipolysis.J. Lipid Res. 2007; 48: 2547-2559Abstract Full Text Full Text PDF PubMed Scopus (757) Google Scholar). Plin1 (the mouse homolog of human PLIN1) is highly abundant in WAT and brown adipose tissue (BAT). Plin2 and Plin3 are expressed in many cell types, whereas Plin4 is abundant in adipose tissue. In contrast, we (4Yamaguchi T. Matsushita S. Motojima K. Hirose F. Osumi T. MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor α.J. Biol. Chem. 2006; 281: 14232-14240Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar) showed that Plin5 is highly concentrated in the heart, whereas other groups (5Wolins N.E. Quaynor B.K. Skinner J.R. Tzekov A. Croce M.A. Gropler M.C. Varma V. Yao-Borengasser A. Rasouli N. Kern P.A. Finck B.N. Bickel P.E. OXPAT/PAT-1 is a PPAR-induced lipid droplet protein that promotes fatty acid utilization.Diabetes. 2006; 55: 3418-3428Crossref PubMed Scopus (246) Google Scholar, 6Dalen K.T. Dahl T. Holter E. Arntsen B. Londos C. Sztalryd C. Nebb H.I. LSDP5 is a PAT protein specifically expressed in fatty acid-oxidizing tissues.Biochim. Biophys. Acta. 2007; 1771: 210-227Crossref PubMed Scopus (185) Google Scholar) demonstrated that this protein is expressed in oxidative tissues, including the heart, BAT, skeletal muscle, and the liver. Plin1 is the best characterized member of the perilipin family, playing a central function in both the storage and catecholamine-dependent mobilization of TAG in WAT (7Martinez-Botas J. Anderson J.B. Tessier D. Lapillonne A. Chang B.H. Quast M.J. Gorenstein D. Chen K.H. Chan L. Absence of perilipin results in leanness and reverses obesity in Lepr(db/db) mice.Nat. Genet. 2000; 26: 474-479Crossref PubMed Scopus (491) Google Scholar, 8Tansey J.T. Sztalryd C. Gruia-Gray J. Roush D.L. Zee J.V. Gavrilova O. Reitman M.L. Deng C.X. Li C. Kimmel A.R. Londos C. Perilipin ablation results in a lean mouse with aberrant adipocyte lipolysis, enhanced leptin production, and resistance to diet-induced obesity.Proc. Natl. Acad. Sci. U.S.A. 2001; 98: 6494-6499Crossref PubMed Scopus (604) Google Scholar). Recent studies (9Sztalryd C. Xu G. Dorward H. Tansey J.T. Contreras J.A. Kimmel A.R. Londos C. Perilipin A is essential for the translocation of hormone-sensitive lipase during lipolytic activation.J. Cell Biol. 2003; 161: 1093-1103Crossref PubMed Scopus (417) Google Scholar, 10Lass A. Zimmermann R. Haemmerle G. Riederer M. Schoiswohl G. Schweiger M. Kienesberger P. Strauss J.G. Gorkiewicz G. Zechner R. Adipose triglyceride lipase-mediated lipolysis of cellular fat stores is activated by CGI-58 and defective in Chanarin-Dorfman syndrome.Cell Metab. 2006; 3: 309-319Abstract Full Text Full Text PDF PubMed Scopus (674) Google Scholar, 11Yamaguchi T. Omatsu N. Morimoto E. Nakashima H. Ueno K. Tanaka T. Satouchi K. Hirose F. Osumi T. CGI-58 facilitates lipolysis on lipid droplets but is not involved in the vesiculation of lipid droplets caused by hormonal stimulation.J. Lipid Res. 2007; 48: 1078-1089Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 12Miyoshi H. Perfield 2nd, J.W. Souza S.C. Shen W.J. Zhang H.H. Stancheva Z.S. Kraemer F.B. Obin M.S. Greenberg A.S. Control of adipose triglyceride lipase action by serine 517 of perilipin A globally regulates protein kinase A-stimulated lipolysis in adipocytes.J. Biol. Chem. 2007; 282: 996-1002Abstract Full Text Full Text PDF PubMed Scopus (224) Google Scholar, 13Shen W.J. Patel S. Miyoshi H. Greenberg A.S. Kraemer F.B. Functional interaction of hormone-sensitive lipase and perilipin in lipolysis.J. Lipid Res. 2009; 50: 2306-2313Abstract Full Text Full Text PDF PubMed Scopus (88) Google Scholar, 14Granneman J.G. Moore H.P. Krishnamoorthy R. Rathod M. Perilipin controls lipolysis by regulating the interactions of AB-hydrolase containing 5 (Abhd5) and adipose triglyceride lipase (Atgl).J. Biol. Chem. 2009; 284: 34538-34544Abstract Full Text Full Text PDF PubMed Scopus (261) Google Scholar, 15Radner F.P. Streith I.E. Schoiswohl G. Schweiger M. Kumari M. Eichmann T.O. Rechberger G. Koefeler H.C. Eder S. Schauer S. Theussl H.C. Preiss-Landl K. Lass A. Zimmermann R. Hoefler G. Zechner R. Haemmerle G. Growth retardation, impaired triacylglycerol catabolism, hepatic steatosis, and lethal skin barrier defect in mice lacking comparative gene identification-58 (CGI-58).J. Biol. Chem. 2010; 285: 7300-7311Abstract Full Text Full Text PDF PubMed Scopus (151) Google Scholar) have presented an elaborate model for the actions of Plin1 involving adipose triacylglycerol lipase (ATGL), comparative gene identification (CGI)-58 (also called α,β-hydrolase domain-containing 5), a coactivator of ATGL, and hormone-sensitive lipase (HSL) (for review see Ref. 16Zechner R. Kienesberger P.C. Haemmerle G. Zimmermann R. Lass A. Adipose triglyceride lipase and the lipolytic catabolism of cellular fat stores.J. Lipid Res. 2009; 50: 3-21Abstract Full Text Full Text PDF PubMed Scopus (404) Google Scholar). The functions of other perilipin family members are less well understood, although the proteins were shown to protect TAG from attack by lipases (17Bell M. Wang H. Chen H. McLenithan J.C. Gong D.W. Yang R.Z. Yu D. Fried S.K. Quon M.J. Londos C. Sztalryd C. Consequences of lipid droplet coat protein down-regulation in liver cells. Abnormal lipid droplet metabolism and induction of insulin resistance.Diabetes. 2008; 57: 2037-2045Crossref PubMed Scopus (150) Google Scholar). Plin2−/− mice are resistant to a high fat diet-induced hepatosteatosis (18Chang B.H. Li L. Paul A. Taniguchi S. Nannegari V. Heird W.C. Chan L. Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.Mol. Cell. Biol. 2006; 26: 1063-1076Crossref PubMed Scopus (256) Google Scholar), and Plin3 compensates for the defect of Plin2 in these mice (19Sztalryd C. Bell M. Lu X. Mertz P. Hickenbottom S. Chang B.H. Chan L. Kimmel A.R. Londos C. Functional compensation for adipose differentiation-related protein (ADFP) by Tip47 in an ADFP null embryonic cell line.J. Biol. Chem. 2006; 281: 34341-34348Abstract Full Text Full Text PDF PubMed Scopus (102) Google Scholar). Some 60–70% of the large energy demand of the heart for contractile function is fulfilled by FA (20van der Vusse G.J. van Bilsen M. Glatz J.F. Cardiac fatty acid uptake and transport in health and disease.Cardiovasc. Res. 2000; 45: 279-293Crossref PubMed Scopus (230) Google Scholar). Thus, the heart has an extremely high capacity of lipid turnover and particularly small LDs. We presumed that Plin5 has a critical role in determining the properties and functions of LDs in the heart, and hence it is the key to understanding the physiological significance of heart LDs. Accordingly, we generated and characterized Plin5 knock-out (Plin5−/−) mice. To our surprise, Plin5−/− mice lacked detectable LDs in the heart, substantiating the essential role of Plin5 in maintaining heart LDs. These mice suffered from an accelerated decline in heart contractile function with age, probably due to increased production of reactive oxygen species (ROS). We propose that LDs contribute to suppressing oxidative stress in the heart by sequestering FA from excessive oxidative metabolism. Plin5−/− mice were produced by using the standard gene disruption procedure (Fig. 1A). The Plin5−/− mice were backcrossed to the C57BL/6J strain (CLEA Japan, Inc.) for four generations. Plin5+/+ and Plin5−/− mice were obtained by mating respective homozygous parents. Mice were housed under standard conditions at 24–26 °C with a 12:12 h light/dark cycle and given free access to standard chow (CLEA Japan, Inc.) and water. For some experiments, mice were fasted overnight by food deprivation. For the treatment with N-acetylcysteine (NAC), the chemical was dissolved in water at 1.88 mg/ml and given to the mice instead of water. The treatment was continued from 16 to 18 weeks of age to the day of experiment at 30–32 weeks of age, replacing the solution every 2–3 days. Assuming that mice 30 g in body weight drink 8 ml of water per day on average (21Bachmanov A.A. Reed D.R. Beauchamp G.K. Tordoff M.G. Food intake, water intake, and drinking spout side preference of 28 mouse strains.Behav. Genet. 2002; 32: 435-443Crossref PubMed Scopus (454) Google Scholar), this dose would correspond to 500 mg of NAC/kg/day. All procedures were performed in accordance with the guidelines established by University of Hyogo for the care and use of experimental animals. Tissues were fixed with 10% formalin/PBS, incubated with 10% sucrose/PBS, and then with 20% sucrose/PBS, each overnight at 4 °C. The tissues were embedded in O.C.T. compound (Tissue-Tek) and sectioned 10 μm thick in a cryostat (Leica CM3050S-III). Sections were air-dried and stored at −80 °C until used. After removal of the O.C.T. compound by washing with water, sections were stained for lipid with 0.18% Oil Red O (ORO) in 60% isopropyl alcohol. Sections were washed with 60% isopropyl alcohol, counterstained with hematoxylin, and subjected to microscopic analysis. Cryosections as prepared above were freed from the O.C.T. compound and permeabilized with methanol for 20 min at −20 °C. After washing with PBS three times for 5 min each at room temperature, sections were blocked with 2% BSA/PBS for 1 h at room temperature. Sections were then incubated with primary antibodies to Plin5 (raised in rabbit (4Yamaguchi T. Matsushita S. Motojima K. Hirose F. Osumi T. MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor α.J. Biol. Chem. 2006; 281: 14232-14240Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar)) and Plin2 (raised in guinea pig) and diluted 1000-fold in 2% BSA/PBS overnight at 4 °C. Secondary antibodies, Alexa 488- and 594-conjugated anti-guinea pig and rabbit IgG (Molecular Probes), respectively, were used at a 500-fold dilution in 2% BSA/PBS for 1 h at room temperature. For double staining of Plin5 and lipid, permeabilization with methanol was omitted to avoid a loss of lipid. Because of freezing and thawing of the cryosections, antibodies were allowed to access intracellular compartments, although the fluorescence signals were inevitably weaker than those obtained by usual permeabilization. Sections were first stained for Plin5 and then with 100 μm Bodipy 493/503 (Molecular Probes). Samples were examined in a confocal laser microscope (Zeiss LSM510). Dissected tissues were prefixed with 2% glutaraldehyde/PBS at 4 °C. After being washed with PBS, tissues were fixed with 2% osmium tetroxide/PBS for 2 h at 4 °C. Specimens were embedded in Quetol-812 (Nissin EM), and then sectioned and observed in a JEM-1200EX (JEOL). Lipids were extracted from tissues according to a standard procedure (22Bligh E.G. Dyer W.J. A rapid method of total lipid extraction and purification.Can. J. Biochem. Physiol. 1959; 37: 911-917Crossref PubMed Scopus (42694) Google Scholar). TAG and FA levels were measured using triglyceride E-test (WAKO) and nonesterified fatty acid C-test (WAKO), respectively. Protein was determined with a protein assay kit (Bio-Rad), using BSA as a standard. Protein extracts were prepared from tissues by using lysis buffer (20 mm Tris-HCl (pH 7.5), 1% Triton X-100, 150 mm NaCl, 10 mm NaF, 1 mm sodium pyrophosphate, 1 mm sodium orthovanadate, protease inhibitor mixture (Roche Applied Science), and 1 mm EDTA). Protein concentrations were quantified, and the extracts were subjected to immunoblotting. Total RNA was prepared from cardiac ventricles using QIAzol reagent (Qiagen) and reverse-transcribed. Target genes were amplified with a SYBR qPCR kit (KAPA Biosystems) and quantified using an ABI PRISM7000 (Applied Biosystems). The hearts from fed and fasted wild-type and Plin5−/− mice were homogenized in 500 μl of 20 mm Hepes-NaOH (pH 7.4) containing protease inhibitor mixture (Roche Applied Science), using a Potter-Elvehjem homogenizer. After the removal of nuclei and cell debris by centrifugation at 700 × g for 10 min, 300 μl of the postnuclear supernatant was mixed with an equal volume of 60% sucrose and placed below a 5-ml 0–30% (w/v) sucrose gradient in a centrifuge tube. Samples were centrifuged for 6 h at a maximal gravity of 140,000 × g at 4 °C in a swinging bucket rotor, S52ST, in a Hitachi CS100GXL ultracentrifuge. Samples were collected into 24 consecutive portions of 230 μl each from the top and combined into eight fractions by mixing three consecutive portions for subsequent analyses. Because a substantial amount of pellet was obtained at the bottom, it was resuspended in 230 μl of 30% sucrose, 10 mm Hepes-NaOH (pH 7.4), and numbered as fraction 9. For immunoblotting, a one-third volume of fraction 9 relative to those of fractions 1–8 was loaded onto gels. In another centrifugal experiment, 0–51.2% (w/v) sucrose gradient was employed. Mice fasted overnight were perfused with 10 ml of 50 μm bromoenol lactone (BEL) in saline for 6 min. The heart was then immediately isolated and analyzed. For the visualization of LDs, Nile Red staining was performed with frozen sections. In vitro lipase assay was performed as described (23Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. Kratky D. Wagner E.F. Klingenspor M. Hoefler G. Zechner R. Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar). Ventricular myocytes were obtained as described previously (24Nakamura T.Y. Goda K. Okamoto T. Kishi T. Nakamura T. Goshima K. Contractile and morphological impairment of cultured fetal mouse myocytes induced by oxygen radicals and oxidants. Correlation with intracellular Ca2+ concentration.Circ. Res. 1993; 73: 758-770Crossref PubMed Scopus (48) Google Scholar). Briefly, ventricular tissues isolated 1.5–3 days after birth were cut into small pieces and digested to produce single cells with an enzyme solution (3:1 mixture of 1 mg/ml collagenase and 0.25% trypsin in PBS). The cells obtained were seeded into collagen-coated 35-mm dishes and incubated in 2 ml of Dulbecco's modified Eagle's medium (DMEM), 10% fetal bovine serum. After incubation overnight, attached cells were subjected to an FA oxidation assay. In each experiment, cardiomyocytes obtained from 3 to 4 mice were seeded into six 35-mm dishes and incubated overnight, as described above. Cells were cultured for a further 2 days in DMEM, 10% fetal bovine serum supplemented with 200 μm oleic acid conjugated with BSA to allow most cells to start spontaneous beating. Cells were then treated with 1 ml of preincubation medium (DMEM, 200 μm oleic acid) with or without 40 μm etomoxir (three dishes each) for 1 h. Subsequently, cells were incubated in 1 ml of the assay medium (DMEM, 20 mm Hepes-NaOH (pH 7.4) containing 4 μm [1-14C]palmitic acid conjugated with α-cyclodextrin), supplemented with or without 40 μm etomoxir for 1 h. During this procedure, each 35-mm dish was uncovered and put in a covered 10-cm dish containing a piece of filter paper soaked in 1 n NaOH to trap radioactive CO2 (“1st filter paper”). After incubation, the culture supernatants were transferred to test tubes, and the cells were dissolved in 300 μl of cell lysis buffer (25 mm Tris-HCl (pH 7.5), 1 mm EDTA, 0.1% Triton X-100). Protein concentrations of the cell lysates were quantified for 50-μl aliquots. One hundred microliters of 10% BSA followed by 150 μl of 3 m perchloric acid were added to the remaining cell lysates, which were then mixed and centrifuged. The supernatants contained intracellular acid-soluble materials (ASM), representing active metabolic intermediates. The pellet was extracted with chloroform/methanol (2:1), and the organic layer was collected (intracellular acid-insoluble fraction, representing cellular lipids). The culture supernatants were also mixed with BSA and perchloric acid and left for 30 min at room temperature, during which time the tubes were covered with parafilm attached with NaOH-soaked filter paper to trap CO2 forced out from the culture supernatants (“2nd filter paper”). The mixtures were then centrifuged and separated into acid-soluble (extracellular ASM, representing dead-end metabolites such as acetic acid) and acid-insoluble (extracellular acid-insoluble fraction, mostly representing unincorporated FA) fractions, as described above. The filter paper and soluble and insoluble fractions were subjected to scintillation counting. β-Oxidation activity was assessed by the radioactivity of CO2 (sum of radioactivities of 1st and 2nd filter papers), intracellular as well as extracellular ASM, with the first two representing metabolites en route to complete oxidation in mitochondria. Incorporation of FA was evaluated from the sum of the radioactivity of CO2, intracellular and extracellular ASM, and the intracellular acid-insoluble fraction. Values obtained for three culture dishes were averaged and used as a result of a single experiment. Blood glucose levels were determined by Glutest sensor (SKK). Serum ketone bodies were determined with a β-hydroxybutyrate assay kit (Cayman). Insulin was quantified using an insulin ELISA kit (Shibayagi). Mice were put in cages individually and allowed to access food and water ad libitum during the experiment. Food intake was measured for 10 days, and average intake per day was calculated. The respiratory gas analysis was performed with an Arco-2000 (Arco System), as described previously (25Matsumura S. Saitou K. Miyaki T. Yoneda T. Mizushige T. Eguchi A. Shibakusa T. Manabe Y. Tsuzuki S. Inoue K. Fushiki T. Mercaptoacetate inhibition of fatty acid β-oxidation attenuates the oral acceptance of fat in BALB/c mice.Am. J. Physiol. Regul. Integr. Comp. Physiol. 2008; 295: R82-R91Crossref PubMed Scopus (17) Google Scholar). Locomotor activity was measured with a 14-channel DAS system (Neuroscience). Heart tissue samples were homogenized in lysis buffer as described above. Protein concentrations were quantified, and the extracts were subjected to a TBARS analysis, according to a published procedure (26Ohkawa H. Ohishi N. Yagi K. Assay for lipid peroxides in animal tissues by thiobarbituric acid reaction.Anal. Biochem. 1979; 95: 351-358Crossref PubMed Scopus (23164) Google Scholar), with slight modifications. For age-matched mice under 1.5% isoflurane anesthesia, heart rate and rhythm were monitored using 4-limb-lead electrocardiography (TMH150, Visual Sonics). Left ventricular contractile forces were quantified by transthoracic echocardiography using Vevo2100 (Visual Sonics). The percentage of left ventricular fractional shortening (FS) was calculated as follows: ((LVID;d − LVID;s)/LVID;d) × 100, where LVID;d and LVID;s indicate left ventricular end-diastolic and end-systolic dimensions, respectively. When the animals were also subjected to a TBARS assay, the hearts were excised several days after the echocardiographic measurement. All data are shown as means ± S.E. Data were analyzed with Student's t test, and differences with p < 0.05 were considered statistically significant. The correlation between heart parameters and TBARS values was assessed by a regression analysis. Plin5−/− mice were generated (Fig. 1, A and B), and the absence of Plin5 mRNA and protein in tissues of mutant mice was confirmed (Fig. 1, C and D). Expression of Plin4, a close neighbor of Plin5 in the genome, was not affected (Fig. 1C). Intercrossing of Plin5+/− mice provided Plin5−/− offspring at a Mendelian ratio. Deletion of Plin5 caused no abnormality in either cumulative body weight or body length (data not shown). Weights of tissues, including the heart, were not significantly different between the two genotypes of mice at 16–20 weeks of age (data not shown). Thus, Plin5 deficiency does not cause apparent defects in growth and development. Further experiments were performed with male mice of age 16–20 weeks, unless otherwise noted. Based on the particularly abundant expression of Plin5 in the heart (4Yamaguchi T. Matsushita S. Motojima K. Hirose F. Osumi T. MLDP, a novel PAT family protein localized to lipid droplets and enriched in the heart, is regulated by peroxisome proliferator-activated receptor α.J. Biol. Chem. 2006; 281: 14232-14240Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar), we expected its deficiency to affect the properties of LDs most prominently in the heart. Accordingly, we first stained the heart sections of wild-type and Plin5−/− mice with ORO (Fig. 2A). In wild-type mice, small LDs were observed in a limited number of heart cells in the fed state. LDs were markedly augmented upon fasting as reported previously (27Suzuki J. Shen W.J. Nelson B.D. Selwood S.P. Murphy Jr., G.M. Kanehara H. Takahashi S. Oida K. Miyamori I. Kraemer F.B. Cardiac gene expression profile and lipid accumulation in response to starvation.Am. J. Physiol. Endocrinol. Metab. 2002; 283: E94-E102Crossref PubMed Scopus (45) Google Scholar), due to increased delivery of FA from WAT. Surprisingly, no LDs were detected in the hearts of Plin5−/− mice by ORO staining either in the fed or fasted state. By electron microscopy, LDs were not observed in the hearts of fasted Plin5−/− mice (Fig. 2B). We also measured the content of TAG and FA in the heart. Consistent with the apparent absence of LDs, the TAG content in the hearts of Plin5−/− mice was lower than that in control mice (Fig. 3A, panel a), in both fed and fasted animals. The FA level was also lower in the hearts of Plin5−/− mice than that of wild-type mice (Fig. 3B, panel a).FIGURE 3TAG and FA contents in the tissues of fed and fasted wild-type and Plin5−/− mice. A, TAG content in the heart (panel a), soleus muscle (panel b), liver (panel c), BAT (panel d), and inguinal WAT (panel e) (n = 5–8 per group). B, free FA content in the heart (panel a), soleus muscle (panel b), and liver (panel c) (n = 5–8 per group). Open bars, wild-type mice, and filled bars, Plin5−/− mice. ***, p < 0.001; **, p < 0.01; *, p < 0.05; ###, p < 0.001; ##, p < 0.01; #, p < 0.05, against fed animals of the same genotypes.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Effect of Plin5 ablation on the contents of TAG and FA was also examined for other tissues. In soleus muscle, TAG content tended to be slightly lower in Plin5−/− mice than in control mice in the fed state, but it" @default.
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• W2084516396 date "2012-07-01" @default.
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• W2084516396 title "Perilipin 5, a Lipid Droplet-binding Protein, Protects Heart from Oxidative Burden by Sequestering Fatty Acid from Excessive Oxidation" @default.
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