Lipoprotein lipase activity is required for cardiac lipid droplet production
2014; Elsevier BV; Volume: 55; Issue: 4 Linguagem: Inglês
10.1194/jlr.m043471
ISSN1539-7262
AutoresChad M. Trent, Shuiqing Yu, Yunying Hu, Nathan Skoller, Lesley A. Huggins, Shunichi Homma, Ira J. Goldberg,
Tópico(s)Muscle metabolism and nutrition
ResumoThe rodent heart accumulates TGs and lipid droplets during fasting. The sources of heart lipids could be either FFAs liberated from adipose tissue or FAs from lipoprotein-associated TGs via the action of lipoprotein lipase (LpL). Because circulating levels of FFAs increase during fasting, it has been assumed that albumin transported FFAs are the source of lipids within heart lipid droplets. We studied mice with three genetic mutations: peroxisomal proliferator-activated receptor α deficiency, cluster of differentiation 36 (CD36) deficiency, and heart-specific LpL deletion. All three genetically altered groups of mice had defective accumulation of lipid droplet TGs. Moreover, hearts from mice treated with poloxamer 407, an inhibitor of lipoprotein TG lipolysis, also failed to accumulate TGs, despite increased uptake of FFAs. TG storage did not impair maximal cardiac function as measured by stress echocardiography. Thus, LpL hydrolysis of circulating lipoproteins is required for the accumulation of lipids in the heart of fasting mice. The rodent heart accumulates TGs and lipid droplets during fasting. The sources of heart lipids could be either FFAs liberated from adipose tissue or FAs from lipoprotein-associated TGs via the action of lipoprotein lipase (LpL). Because circulating levels of FFAs increase during fasting, it has been assumed that albumin transported FFAs are the source of lipids within heart lipid droplets. We studied mice with three genetic mutations: peroxisomal proliferator-activated receptor α deficiency, cluster of differentiation 36 (CD36) deficiency, and heart-specific LpL deletion. All three genetically altered groups of mice had defective accumulation of lipid droplet TGs. Moreover, hearts from mice treated with poloxamer 407, an inhibitor of lipoprotein TG lipolysis, also failed to accumulate TGs, despite increased uptake of FFAs. TG storage did not impair maximal cardiac function as measured by stress echocardiography. Thus, LpL hydrolysis of circulating lipoproteins is required for the accumulation of lipids in the heart of fasting mice. The human heart will accumulate TGs in lipid droplets in disease states such as obesity and diabetes. Whether TG storage directly leads to reduced heart function, i.e., lipotoxicity (1Abel E.D. O'Shea K.M. Ramasamy R. Insulin resistance: metabolic mechanisms and consequences in the heart.Arterioscler. Thromb. Vasc. Biol. 2012; 32: 2068-2076Crossref PubMed Scopus (150) Google Scholar, 2Goldberg I.J. Trent C.M. Schulze P.C. Lipid metabolism and toxicity in the heart.Cell Metab. 2012; 15: 805-812Abstract Full Text Full Text PDF PubMed Scopus (353) Google Scholar), or is a marker for accumulation of other toxic lipids is unclear. Evidence suggesting that stored TGs in cardiomyocytes are not always toxic has come from experiments in genetically modified mice. For instance, overexpression of the final enzyme in TG synthesis, diacylglycerol acyltransferase (DGAT)1, in cardiomyocytes increased TG stores but reduced accumulation of toxic lipids and did not reduce heart function (3Liu L. Shi X. Bharadwaj K.G. Ikeda S. Yamashita H. Yagyu H. Schaffer J.E. Yu Y.H. Goldberg I.J. DGAT1 expression increases heart triglyceride content but ameliorates lipotoxicity.J. Biol. Chem. 2009; 284: 36312-36323Abstract Full Text Full Text PDF PubMed Scopus (175) Google Scholar). Overexpression of DGAT1 in skeletal muscle also increased TG storage in mice with diet-induced obesity and mimicked the "athlete's paradox" observed in endurance-trained humans; skeletal muscle DGAT1 transgenic mice had increased FA oxidation and improved insulin sensitivity (4Liu L. Shi X. Choi C.S. Shulman G.I. Klaus K. Nair K.S. Schwartz G.J. Zhang Y. Goldberg I.J. Yu Y.H. Paradoxical coupling of triglyceride synthesis and fatty acid oxidation in skeletal muscle overexpressing DGAT1.Diabetes. 2009; 58: 2516-2524Crossref PubMed Scopus (52) Google Scholar). In contrast, increased TG accumulation in the human heart correlates with reduced heart function (5Marfella R. Di Filippo C. Portoghese M. Barbieri M. Ferraraccio F. Siniscalchi M. Cacciapuoti F. Rossi F. D'Amico M. Paolisso G. Myocardial lipid accumulation in patients with pressure-overloaded heart and metabolic syndrome.J. Lipid Res. 2009; 50: 2314-2323Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 6Sharma S. Adrogue J.V. Golfman L. Uray I. Lemm J. Youker K. Noon G.P. Frazier O.H. Taegtmeyer H. Intramyocardial lipid accumulation in the failing human heart resembles the lipotoxic rat heart.FASEB J. 2004; 18: 1692-1700Crossref PubMed Scopus (586) Google Scholar). Moreover, greater TG stores are often associated with greater FA oxidation and greater injury during ischemia/reperfusion in isolated perfused hearts (7Lopaschuk G.D. Ussher J.R. Folmes C.D. Jaswal J.S. Stanley W.C. Myocardial fatty acid metabolism in health and disease.Physiol. Rev. 2010; 90: 207-258Crossref PubMed Scopus (1396) Google Scholar). Thus, the role of TG stores in the heart is unclear. Even the substrate used for heart TG production has not been established. One physiologic stimulus that causes lipid accumulation in mouse hearts is prolonged fasting (8Suzuki 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). Because starvation is a threat to survival, lipid accumulation in the heart may be an adaptation to accommodate future energetic demands, to protect the heart from lipotoxicity, or to do both. Understanding the features of this adaptation may provide insight into mechanisms that drive lipid accumulation under physiologic and pathological conditions. During fasting, animals rely exclusively on stored energy. While the liver produces and releases glucose under fasting conditions, this is insufficient to meet the energetic demands of the body (9Cahill Jr, G.F. Fuel metabolism in starvation.Annu. Rev. Nutr. 2006; 26: 1-22Crossref PubMed Scopus (708) Google Scholar). Adipose tissue is the major storage depot for energy in the form of TGs. During the fed state, dietary glucose stimulates insulin secretion, which simultaneously promotes glucose utilization and lipid storage. During fasting, circulating insulin levels fall while glucagon and catecholamines increase. This shift in the hormonal milieu leads to an activation of glycogenolysis in the skeletal muscle and liver and lipolysis in the adipose tissue. However, prolonged fasting will deplete glycogen stores and thus the energy demands of peripheral tissues must rely on both the liver, to secrete glucose and ketone bodies, and TGs and adipose tissue, to secrete FFAs and glycerol. Adipose tissue secreted glycerol, as well as lactate secreted from both adipose tissue and muscle, are taken up by the liver and used as substrates for gluconeogenesis. We tested to determine whether reduced FA oxidation increased fasting-induced TG accumulation in the heart. To do this we studied PPARα knockout mice. Surprisingly, we found that fasted Ppara−/− mice had no lipid droplet accumulation in hearts and had a marked reduction in mRNA levels of the FA transporter cluster of differentiation 36 (CD36) as well as lipoprotein lipase (LpL) (10Kazantzis M. Stahl A. Fatty acid transport proteins, implications in physiology and disease.Biochim. Biophys. Acta. 2012; 1821: 852-857Crossref PubMed Scopus (172) Google Scholar, 11Glatz J.F. Luiken J.J. Bonen A. Membrane fatty acid transporters as regulators of lipid metabolism: implications for metabolic disease.Physiol. Rev. 2010; 90: 367-417Crossref PubMed Scopus (516) Google Scholar); LpL is required for heart uptake of FFAs from lipoprotein TGs. We then assessed the specific roles of CD36 and LpL in heart TG accumulation. Our data show that LpL activity is required for the accumulation of heart lipid droplets. In addition, we demonstrated that lipid droplet accumulation does not affect maximal systolic function of the heart. We used 3–4-month-old male C57BL/6 mice, Ppara−/− mice (12Leone T.C. Weinheimer C.J. Kelly D.P. A critical role for the peroxisome proliferator-activated receptor alpha (PPARalpha) in the cellular fasting response: the PPARalpha-null mouse as a model of fatty acid oxidation disorders.Proc. Natl. Acad. Sci. USA. 1999; 96: 7473-7478Crossref PubMed Scopus (819) Google Scholar), Cd36−/− mice (13Febbraio M. Abumrad N.A. Hajjar D.P. Sharma K. Cheng W. Pearce S.F. Silverstein R.L. A null mutation in murine CD36 reveals an important role in fatty acid and lipoprotein metabolism.J. Biol. Chem. 1999; 274: 19055-19062Abstract Full Text Full Text PDF PubMed Scopus (655) Google Scholar), floxed LpL mice (LpLflox/flox), and heart-specific LpL knockout (hLpL0) mice (14Noh H.L. Okajima K. Molkentin J.D. Homma S. Goldberg I.J. Acute lipoprotein lipase deletion in adult mice leads to dyslipidemia and cardiac dysfunction.Am. J. Physiol. Endocrinol. Metab. 2006; 291: E755-E760Crossref PubMed Scopus (43) Google Scholar). Mice were raised on a normal chow diet. C57BL/6 mice were used as controls for both Ppara−/− and Cd36−/− mice and LpLflox/flox littermates served as controls for the hLpL0 studies. Mice of each genotype were divided into two groups. One group was subjected to a 16 h overnight fast and the other group was fed ad libitum over the same time period. These mice were then euthanized with a lethal injection of 100 mg/kg ketamine and 10 mg/kg xylazine. All procedures were approved by the Columbia University Institutional Animal Care and Use Committee. A ventral incision was made after administration of ketamine-xylazine. The left ventricle of the heart was perfused with 10 ml of PBS or until the liver appeared blanched. Tissues were rapidly removed and frozen in liquid nitrogen. Heart pieces were embedded into Tissue-Tek OCT compound (Sakura) for histology. Two hundred microliters of blood were drawn from each animal and then centrifuged at 2,000 rpm for 10 min to obtain plasma. Plasma was utilized for measurement of TGs, FFAs, and glucose by colorimetric assays. TG measurements were made using the Thermo Scientific Infinity assay (Thermo Scientific), FFAs were measured using the Wako NEFA kit, and plasma glucose was measured using the Wako Autokit Glucose kit (Wako Life Sciences). The lipid extraction protocol was adapted from the Folch method (15Folch J. Lees M. Sloane Stanley G.H. A simple method for the isolation and purification of total lipides from animal tissues.J. Biol. Chem. 1957; 226: 497-509Abstract Full Text PDF PubMed Google Scholar). Approximately 100 mg of tissue in 1 ml of PBS were homogenized using stainless steel beads for 1 min in a bead beater homogenizer. From each sample, 50 μl were removed for protein analysis and 3 ml of 2:1 chloroform:methanol was added to the rest and vortexed. Samples were then centrifuged for 10 min at 3,000 rpm at 4°C. The lower organic phase was then collected and dried under nitrogen gas. The dried lipid was then dissolved in 500 μl of 1 Triton X-100 in chloroform, further dried, and then dissolved in 100 μl of double distilled water. The sample of tissue lysate retained from the lipid extraction protocol was assayed for protein content using Bradford reagent (Bio-Rad) following the instructions of the manufacturer. Using the tissue lipid extract, assays for TGs and FFAs were performed as previously described for plasma lipids. Lipid measurements were normalized to protein content or tissue weight. Heart pieces were embedded in Tissue-Tek OCT compound (Sakura) and then air dried and fixed with formalin. Sections were washed with distilled water and isopropanol. Lipids were then stained with Oil Red O for 18 min, washed with isopropanol and distilled water, and then counterstained with hematoxylin. Slides were once again washed with distilled water and covered with clear nail polish. Images were taken using a Leica DMLB microscope and digital camera. Representative images obtained from five animals of each genotype are shown. Periodic acid-Schiff (PAS) reagent staining was used to demonstrate heart glycogen. Sections of OCT embedded hearts were placed in 10% neutral buffered formalin. Ventricular tissue sections were fixed in methanol for 10 min and stained with PAS reagent (Poly Scientific), hematoxylin, and eosin. Images were taken using a Leica DMLB microscope and digital camera. Four to five mouse hearts were used for each genotype and for each feeding condition, and several representative images were captured for each mouse. Glycogen was also measured by extracting total insoluble carbohydrates and digesting with amyloglucosidase; free glucose was then measured and reported as ratio to tissue weight used for measurement as previously described (16Suzuki Y. Lanner C. Kim J.H. Vilardo P.G. Zhang H. Yang J. Cooper L.D. Steele M. Kennedy A. Bock C.B. et al.Insulin control of glycogen metabolism in knockout mice lacking the muscle-specific protein phosphatase PP1G/RGL.Mol. Cell. Biol. 2001; 21: 2683-2694Crossref PubMed Scopus (129) Google Scholar). Ventricular tissue was hydrolyzed in 300 μl of 5.4 M KOH in a 100°C water bath for 30 min. Then, 100 μl 1 M Na2SO4 and 800 μl of 100% ethanol were added to each sample. Samples were boiled for 5 min and then centrifuged at 10,000 g for 5 min. The glycogen pellet was dissolved in 200 μl water and ethanol precipitation was performed twice with addition of 800 μl of 100% ethanol. Finally, the glycogen pellet was dissolved in 200 μl of 60 U/ml amyloglucosidase (Sigma) in 0.2 M sodium acetate (pH 4.8) and incubated for 3 h at 40°C. Each sample was diluted five times and glucose concentration was measured using the Wako Autokit Glucose kit (Wako Life Sciences). Total RNA was purified from a 30–50 mg piece of heart using TRIzol reagent (Invitrogen) according to the instructions of the manufacturer. cDNA was synthesized using the SuperScript III First-Strand Synthesis SuperMix (Invitrogen) and quantitative real-time PCR was performed with SYBR Green PCR Core reagents (Agilent Technologies) using an Mx3000 sequence detection system (Stratagene, La Jolla, CA). Genes of interest were normalized against 18s rRNA. Primer sequences are listed in supplementary Table I. Hearts were excised as previously described. Approximately 20 mg of tissue was homogenized in RIPA buffer containing protease inhibitor cocktail (Sigma-Aldrich). Twenty-five micrograms of protein extract was applied to SDS-PAGE and transferred onto polyvinylidene fluoride membranes. Antibodies for perilipin (PLIN)2 and PLIN5 were obtained from Santa Cruz Biotechnology (PLIN2, β-actin) and American Research Products (PLIN5). Band density measurements were made using ImageJ software. PLIN2 and PLIN5 band densities were normalized to β-actin band density. Echocardiography was performed on 3–4-month-old male Cd36+/+ (wild-type), Cd36−/−, LpLflox/flox, and hLpL0 mice fasted for 16 h. Two-dimensional echocardiography was performed using a high-resolution imaging system with a 30 MHz imaging transducer (Vevo 770; VisualSonics) in unconscious mice. The mice were anesthetized with 1.5–2% isoflurane and thereafter maintained on 0.5% isoflurane throughout the procedure. Care was taken to minimize sedation by monitoring the heart rate of the mice. Two-dimensional echocardiographic images were obtained using short-axis views at the level of papillary muscles, and each parameter was measured using M-mode view. Images were analyzed offline by a researcher blinded to the murine genotype. Left ventricular end-diastolic dimension (LVEDd) and left ventricular end-systolic dimension (LVEDs) were measured. Percentage fractional shortening (FS), which quantifies contraction of the ventricular wall and is an indication of muscle function, was calculated as FS = ([LVEDd − LVEDs]/LVEDd) × 100. To assess stress response, 0.3 mg/kg isoproterenol (Sigma-Aldrich) was administered intraperitoneally. Successful administration of drug was confirmed by observation of increased heart rate. Poloxamer 407 (P407) was prepared in PBS as previously described (17Millar J.S. Cromley D.A. McCoy M.G. Rader D.J. Billheimer J.T. Determining hepatic triglyceride production in mice: comparison of poloxamer 407 with Triton WR-1339.J. Lipid Res. 2005; 46: 2023-2028Abstract Full Text Full Text PDF PubMed Scopus (199) Google Scholar). Mice were injected intraperitoneally with 1 mg/g body weight of P407 and then fasted for 16 h. Control mice were injected with an equivalent volume of PBS. Mice were euthanized and analyzed as previously described. FFA and glucose uptake were assessed in mice that were injected with PBS or P407 and then fasted for 16 h. [9,10-3H(N)]oleate (PerkinElmer Life Sciences) was complexed to 6% FA-free BSA (Sigma). Mice were injected intravenously with 1 μCi [9,10-3H(N)]oleate-BSA and blood was collected at 0.5, 1, 3, and 5 min after injection. Five minutes after injection, the body cavity was perfused with 10 ml of PBS by cardiac puncture and tissues were excised. Tissue was homogenized in PBS and radioactive counts were measured. Basal glucose uptake was measured in hearts following an intravenous administration of 2.5 μCi of 2-deoxy-D-[1-14C]glucose (PerkinElmer Life Sciences). Blood was collected 2, 30, and 60 min following injection. At 60 min, hearts were perfused with PBS, tissues were excised, and radioactive counts were measured. For all turnover studies, radioactivity per gram of tissue was normalized to the respective 30 s or 2 min plasma counts (injected dose). Data are expressed as mean ± SE. Data were analyzed by the use of unpaired Student's t-test or two-way ANOVA. We first assessed heart lipid storage in Ppara−/− mice. We fasted Ppara+/+ (wild-type) and Ppara−/− mice overnight for 16 h. Fasting increased plasma FFAs 2-fold in Ppara+/+ mice and 3-fold in Ppara−/− mice, but had no significant effect on plasma TGs (Fig. 1A). Plasma glucose decreased approximately 30% in fasted Ppara+/+ mice and approximately 60% in fasted Ppara−/− mice. Fasting increased heart TGs 5-fold in Ppara+/+ mice, but there was no significant TG accumulation in Ppara−/− mice (Fig. 1B). Heart FA levels increased approximately 30% in Ppara+/+ mice, but were not increased in Ppara−/− mice (Fig. 1B). Fasted Ppara+/+ mice had increased Oil Red O staining, but Ppara−/− mice had minimal staining (Fig. 1C). We then assessed heart glycogen storage in Ppara−/− mice to determine whether these hearts depleted their stored carbohydrate. There was no difference in PAS reagent staining of glycogen (Fig. 1D) or extracted glycogen content (Fig. 1E) after fasting. There tended to be increased glycogen in Ppara−/− hearts both before and after fasting. Changes in genes required for lipid and glucose metabolism were determined in hearts of fed and fasted mice. Adipose TG lipase (Atgl), the rate limiting enzyme for intracellular TG lipolysis (18Haemmerle G. Lass A. Zimmermann R. Gorkiewicz G. Meyer C. Rozman J. Heldmaier G. Maier R. Theussl C. Eder S. et al.Defective lipolysis and altered energy metabolism in mice lacking adipose triglyceride lipase.Science. 2006; 312: 734-737Crossref PubMed Scopus (1011) Google Scholar), was decreased by 50% in both fed and fasted Ppara−/− mice compared with Ppara+/+ mice (Fig. 1F). Expression of carnitine palmitoyl transferase (Cpt)1b, the rate limiting enzyme for mitochondrial lipid oxidation, was minimal in both fed and fasted Ppara−/− hearts. Surprisingly, mRNA of acyl CoA oxidase (Acox)1, the first enzyme in the lipid oxidation pathway, was increased in fasted Ppara−/− hearts. However, decreased FA oxidation has been previously observed in these hearts (19Watanabe K. Fujii H. Takahashi T. Kodama M. Aizawa Y. Ohta Y. Ono T. Hasegawa G. Naito M. Nakajima T. et al.Constitutive regulation of cardiac fatty acid metabolism through peroxisome proliferator-activated receptor alpha associated with age-dependent cardiac toxicity.J. Biol. Chem. 2000; 275: 22293-22299Abstract Full Text Full Text PDF PubMed Scopus (266) Google Scholar, 20Campbell F.M. Kozak R. Wagner A. Altarejos J.Y. Dyck J.R. Belke D.D. Severson D.L. Kelly D.P. Lopaschuk G.D. A role for peroxisome proliferator-activated receptor alpha (PPARalpha) in the control of cardiac malonyl-CoA levels: reduced fatty acid oxidation rates and increased glucose oxidation rates in the hearts of mice lacking PPARalpha are associated with higher concentrations of malonyl-CoA and reduced expression of malonyl-CoA decarboxylase.J. Biol. Chem. 2002; 277: 4098-4103Abstract Full Text Full Text PDF PubMed Scopus (222) Google Scholar). Therefore, absence of TG stores was not likely due to increased FA oxidation. As expected, mRNA levels of lipid droplet protein genes Plin2 and Plin5 were minimal in both fed and fasted Ppara−/− mice compared with Ppara+/+ mice (21Yamaguchi 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 alpha.J. Biol. Chem. 2006; 281: 14232-14240Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Fasting dramatically increased expression of pyruvate dehydrogenase kinase (Pdk)4, the negative regulator of glucose oxidation, in hearts from Ppara+/+ mice. Fasted Ppara−/− mice also had increased Pdk4 mRNA levels compared with fed Ppara−/− mice, but these levels were still reduced compared with the Ppara+/+ counterparts. Fasted Ppara−/− mice had increased mRNA expression of glucose transporter (Glut)1, the insulin insensitive glucose transporter, compared with fasted Ppara+/+ hearts, but there was no difference in expression of Glut4, the insulin sensitive glucose transporter. Most remarkable was that fed and fasted Ppara−/− hearts had an ∼80% reduction in lipid uptake genes Cd36 and Lpl (Fig. 1F). We assessed heart mRNA levels of several genes involved in both de novo lipogenesis and TG formation. Expression of acetyl-CoA carboxylase (Acc)2, which is rate-limiting for de novo lipogenesis, was increased in the hearts of fed Ppara−/− mice (supplementary Fig. IIIA). mRNA expression of Fasn, the second rate-limiting enzyme for de novo lipogenesis, was increased in the hearts of both fed and fasted Ppara−/− mice. mRNA levels of Dgat1, the rate limiting enzyme for TG synthesis, were decreased in hearts from both fed and fasted Ppara−/− mice. We also measured gene expression of FA transporters other than Cd36. Hearts from both fed and fasted Ppara−/− mice had decreased expression of FA transport protein 1 (Slc27a1, or Fatp1), but increased expression of FA binding protein-plasma membrane (Got2, or FABPpm). Finally, we measured TG lipase activity in hearts of fed and fasted Ppara+/+ and Ppara−/− mice. Both fed and fasted Ppara−/− mice had 80–90% increased heart TG lipase activity compared with Ppara+/+ mice of the same nutritional status (supplementary Fig. IVA). Next, we determined whether CD36 deficiency would be sufficient to prevent heart TG accumulation during the fasted state. Fasting increased plasma FFAs 3-fold in Cd36−/− mice (Fig. 2A). Plasma TGs tended to be higher in fasted Cd36−/− mice compared with Cd36+/+ mice. Fasted Cd36−/− mice had an ∼50% decrease in plasma glucose. There was no significant TG accumulation in Cd36−/− hearts (Fig. 2B, C). Heart FA content also did not increase in hearts from Cd36−/− mice (Fig. 2B). Glycogen content was similar in all hearts (Fig. 2D, E). Finally, Cd36−/− mouse hearts tended to have decreased expression of lipid metabolism genes (Atgl, Cpt1b, Acox1, Atgl, Plin2, and Plin5) in the fed state, but fasted Cd36−/− mouse hearts had similar gene expression to hearts of Cd36+/+ mice (Fig. 2F). LpL mRNA levels were comparable to control mice in both fasting and fed hearts. Glucose oxidation and uptake genes (Pdk4, Glut1, and Glut4) were comparable between genotypes and feeding conditions. Fed and fasted Cd36−/− mice had decreased expression of Dgat2 (supplementary Fig. IIIB). Hearts of fasted Cd36−/− mice had increased expression of Slc27a1, and both fed and fasted hearts had decreased expression of FA binding protein (Fabp)3. There were no differences in TG lipase activity between feeding conditions or genotype (supplementary Fig. IVB). If circulating FFAs are the source of heart TG stores during fasting, we would expect that loss of lipoprotein TG hydrolysis in the heart would not affect lipid droplet accumulation during fasting. To test this, we fasted hLpL0 mice and compared them to LpLflox/flox littermates. After fasting hLpL0 mice had normal increases in plasma FFA levels, an approximately 2-fold increase (Fig. 3A). hLpL0 mice tended to have slightly elevated TGs in the fasted state, as has been reported (22Augustus A. Yagyu H. Haemmerle G. Bensadoun A. Vikramadithyan R.K. Park S.Y. Kim J.K. Zechner R. Goldberg I.J. Cardiac-specific knock-out of lipoprotein lipase alters plasma lipoprotein triglyceride metabolism and cardiac gene expression.J. Biol. Chem. 2004; 279: 25050-25057Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Plasma glucose fell 20% in both LpLflox/flox and hLpL0 mice. Surprisingly, hLpL0 mice did not accumulate cardiomyocyte TGs during fasting (Fig. 3B, C). Glycogen content was similar in all hearts (Fig. 3D, E). We predicted that changes in gene expression in the fasting hLpL0 hearts would explain the lack of TG stores. mRNA levels of Atgl, Cpt1b, and Acox1 were reduced in both fed and fasted hLpL0 mouse hearts (Fig. 3F), consistent with the reduction in FA oxidation in these hearts (23Augustus A.S. Buchanan J. Park T.S. Hirata K. Noh H.L. Sun J. Homma S. D'Armiento J. Abel E.D. Goldberg I.J. Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction.J. Biol. Chem. 2006; Abstract Full Text Full Text PDF Scopus (104) Google Scholar). hLpL0 mice also had decreased expression of Plin2 and Plin5 compared with fasted LpLflox/flox mice. Although these hearts do not have reduced FFA uptake, Cd36 mRNA was reduced in both fed and fasted hLpL0 hearts. Pdk4 expression was reduced in both fed and fasted hLpL0 hearts compared with LpLflox/flox hearts. Although glucose uptake was increased in hLpL0 hearts (23Augustus A.S. Buchanan J. Park T.S. Hirata K. Noh H.L. Sun J. Homma S. D'Armiento J. Abel E.D. Goldberg I.J. Loss of lipoprotein lipase-derived fatty acids leads to increased cardiac glucose metabolism and heart dysfunction.J. Biol. Chem. 2006; Abstract Full Text Full Text PDF Scopus (104) Google Scholar), fasted Glut1 mRNA levels were lower than controls and did not differ between fed and fasted hLpL0 mouse hearts. Glut4 was also reduced in both fed and fasted hLpL0 hearts. Hearts from both fed and fasted hLpL0 mice had decreased Dgat1 mRNA expression (supplementary Fig. IIIC). Fasted hLpL0 mice had less Dgat2 mRNA expression compared with fasted LpLflox/flox mice. mRNA levels of the rate-limiting enzyme for production of monounsaturated FAs, stearoyl-CoA desaturase (Scd)1, were decreased in hearts of fasted hLpL0 mice. Expression of Slc27a1 was increased in fasted floxed LpL mouse hearts, but not as much in fasted hLpL0 mouse hearts. FA transport protein 6 (Fatp6), Fabp3, and Got2 were all decreased in hearts of fasted hLpL0 mice compared with fasted floxed LpL mice. There were no differences in TG lipase activity between feeding conditions or genotype (supplementary Fig. IVC). A reduction in PLIN2 prevents lipid accumulation in the liver (24Chang 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, 25McManaman J.L. Bales E.S. Orlicky D.J. Jackman M. MacLean P.S. Cain S. Crunk A.E. Mansur A. Graham C.E. Bowman T.A. et al.Perilipin-2-null mice are protected against diet-induced obesity, adipose inflammation, and fatty liver disease.J. Lipid Res. 2013; 54: 1346-1359Abstract Full Text Full Text PDF PubMed Scopus (143) Google Scholar), and we noticed a reduction in Plin2 mRNA. For this reason, we measured PLIN2 protein in the hearts of fed and fasted mice. Fasting increased PLIN2 protein in Cd36+/+ mice, and unexpectedly also in Cd36−/− mouse hearts (Fig. 4A). Band density measurement indicated about a 40–50% increase in PLIN2 after fasting in both Cd36+/+ and Cd36−/− mice (Fig. 4B). Because PLIN5 blocks TG lipolysis (26Pollak N.M. Schweiger M. Jaeger D. Kolb D. Kumari M. Schreiber R. Kolleritsch S. Markolin P. Grabner G.F. 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