Ceramide is a cardiotoxin in lipotoxic cardiomyopathy
2008; Elsevier BV; Volume: 49; Issue: 10 Linguagem: Inglês
10.1194/jlr.m800147-jlr200
ISSN1539-7262
AutoresTae‐Sik Park, Yunying Hu, Hye Lim Noh, Konstantinos Drosatos, Kazue Okajima, Jonathan Buchanan, Joseph Tuinei, Shunichi Homma, Xian-Cheng Jiang, E. Dale Abel, Ira J. Goldberg,
Tópico(s)Metabolism, Diabetes, and Cancer
ResumoCeramide is among a number of potential lipotoxic molecules that are thought to modulate cellular energy metabolism. The heart is one of the tissues thought to become dysfunctional due to excess lipid accumulation. Dilated lipotoxic cardiomyopathy, thought to be the result of diabetes and severe obesity, has been modeled in several genetically altered mice, including animals with cardiac-specific overexpression of glycosylphosphatidylinositol (GPI)-anchored human lipoprotein lipase (LpLGPI). To test whether excess ceramide was implicated in cardiac lipotoxicity, de novo ceramide biosynthesis was inhibited pharmacologically by myriocin and genetically by heterozygous deletion of LCB1, a subunit of serine palmitoyltransferase (SPT). Inhibition of SPT, a rate-limiting enzyme in ceramide biosynthesis, reduced fatty acid and increased glucose oxidation in isolated perfused LpLGPI hearts, improved systolic function, and prolonged survival rates. Our results suggest a critical role for ceramide accumulation in the pathogenesis of lipotoxic cardiomyopathy. Ceramide is among a number of potential lipotoxic molecules that are thought to modulate cellular energy metabolism. The heart is one of the tissues thought to become dysfunctional due to excess lipid accumulation. Dilated lipotoxic cardiomyopathy, thought to be the result of diabetes and severe obesity, has been modeled in several genetically altered mice, including animals with cardiac-specific overexpression of glycosylphosphatidylinositol (GPI)-anchored human lipoprotein lipase (LpLGPI). To test whether excess ceramide was implicated in cardiac lipotoxicity, de novo ceramide biosynthesis was inhibited pharmacologically by myriocin and genetically by heterozygous deletion of LCB1, a subunit of serine palmitoyltransferase (SPT). Inhibition of SPT, a rate-limiting enzyme in ceramide biosynthesis, reduced fatty acid and increased glucose oxidation in isolated perfused LpLGPI hearts, improved systolic function, and prolonged survival rates. Our results suggest a critical role for ceramide accumulation in the pathogenesis of lipotoxic cardiomyopathy. Increasing caloric intake and greater body mass index produce a series of diseases due to tissue lipid accumulation. As storage capacity of adipocytes is exceeded, fat begins to infiltrate the liver, skeletal muscle, and heart (1Zhou Y.T. Grayburn P. Karim A. Shimabukuro M. Higa M. Baetens D. Orci L. Unger R.H. Lipotoxic heart disease in obese rats: implications for human obesity.Proc. Natl. Acad. Sci. USA. 2000; 97: 1784-1789Crossref PubMed Scopus (1056) Google Scholar, 2Molavi B. Rasouli N. Kern P.A. The prevention and treatment of metabolic syndrome and high-risk obesity.Curr. Opin. Cardiol. 2006; 21: 479-485Crossref PubMed Scopus (22) Google Scholar). 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Siesky A. et al.Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.Cell Metab. 2007; 5: 167-179Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar) compared the effects of soybean- and lard-based emulsion infusions and suggested that the two types of lipids cause insulin resistance via different mechanisms. Only saturated fat increased tissue ceramide levels. Genetic and pharmacologic inhibition of the de novo ceramide biosynthetic pathway ameliorated insulin resistance. Aside from ceramide, DAG and/or FA could be responsible for dilated lipotoxic cardiomyopathy. DAG activates protein kinase C in skeletal muscle and aorta, which is associated with insulin resistance (11Griffin M.E. Marcucci M.J. Cline G.W. Bell K. Barucci N. Lee D. Goodyear L.J. Kraegen E.W. White M.F. Shulman G.I. Free fatty acid-induced insulin resistance is associated with activation of protein kinase C theta and alterations in the insulin signaling cascade.Diabetes. 1999; 48: 1270-1274Crossref PubMed Scopus (956) Google Scholar, 12Xia P. Inoguchi T. Kern T.S. Engerman R.L. Oates P.J. King G.L. Characterization of the mechanism for the chronic activation of diacylglycerol-protein kinase C pathway in diabetes and hypergalactosemia.Diabetes. 1994; 43: 1122-1129Crossref PubMed Scopus (409) Google Scholar, 13Avignon A. Yamada K. Zhou X. Spencer B. Cardona O. Saba-Siddique S. Galloway L. Standaert M.L. Farese R.V. Chronic activation of protein kinase C in soleus muscles and other tissues of insulin-resistant type II diabetic Goto-Kakizaki (GK), obese/aged, and obese/Zucker rats. A mechanism for inhibiting glycogen synthesis.Diabetes. 1996; 45: 1396-1404Crossref PubMed Scopus (139) Google Scholar). 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Merkel M. et al.Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.J. Clin. Invest. 2003; 111: 419-426Crossref PubMed Scopus (295) Google Scholar). These hearts have an accumulation of ceramide. In this report, we show that ceramide accumulation plays a significant role in the progression of the dilated cardiomyopathy seen in LpLGPI mice. Using pharmacologic and genetic methods, we tested whether inhibition of SPT in normal and lipotoxic hearts altered cardiac ceramide concentrations and cardiac substrate utilization. Reduction of ceramide was associated with improved cardiac function, reversal of abnormal substrate use, and improved survival. Generation of LpLGPI transgenic mice has been described previously (19Yagyu H. Chen G. Yokoyama M. Hirata K. Augustus A. Kako Y. Seo T. Hu Y. Lutz E.P. Merkel M. et al.Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.J. Clin. Invest. 2003; 111: 419-426Crossref PubMed Scopus (295) Google Scholar). LpLGPI and wild-type (WT) C57BL/6J mice (8–10 weeks old) were fed either a chow diet (Research Diets, New Brunswick, NJ) or chow mixed with myriocin (Sigma Adrich, St. Louis, MO, prepared by Research Diets)(0.3 mg/kg/day) for 6 weeks. Heterozygous LCB1 knockout (LCB1+/−) and LCB1+/− crossed with LpLGPI transgenic mice were fed a normal chow diet. For survival analyses, the same dose of myriocin was fed to 12 week-old LpLGPI (n = 14) and C57Bl/6J mice (n = 14) for 40 weeks. All animals were maintained on a 12 h light-dark cycle. Generation of a human cardiomyocyte cell line, AC16, has been described previously by Davidson et al. (20Davidson M.M. Nesti C. Palenzuela L. Walker W.F. Hernandez E. Protas L. Hirano M. Isaac N.D. Novel cell lines derived from adult human ventricular cardiomyocytes.J. Mol. Cell. Cardiol. 2005; 39: 133-147Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar). Cells were grown in DMEM/F12 media containing 12.5% FBS and incubated at 37°C in an atmosphere containing 5% CO2/95% air. Cardiomyocytes that were 70–80% confluent were switched to DMEM/F12 media containing 1% FBS and treated with various concentrations of palmitic acid conjugated with 2% FA-free BSA in the presence or absence of 1 μM myriocin for 16 h. In other experiments, cells were treated with C6-ceramide for 16 h after incubation in DMEM/F12 containing 2% FA-free BSA. mRNA for RT-PCR was isolated using TRizol (Invitrogen). Cardiac metabolism was measured in hearts isolated from 14 to 16 week-old male WT and LpLGPI mice (n = 5 per genotype). All hearts were prepared and perfused in the working mode, using protocols that have been previously described (21Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice.Am. J. Physiol. Endocrinol. Metab. 2000; 279: E1104-E1113Crossref PubMed Google Scholar, 22Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter.Am. J. Physiol. Endocrinol. Metab. 2001; 280: E420-E427Crossref PubMed Google Scholar, 23Belke D.D. Larsen T.S. Lopaschuk G.D. Severson D.L. Glucose and fatty acid metabolism in the isolated working mouse heart.Am. J. Physiol. 1999; 277: R1210-R1217Crossref PubMed Google Scholar, 24Mazumder P.K. O'Neill B.T. Roberts M.W. Buchanan J. Yun U.J. Cooksey R.C. Boudina S. Abel E.D. Impaired cardiac efficiency and increased fatty acid oxidation in insulin-resistant ob/ob mouse hearts.Diabetes. 2004; 53: 2366-2374Crossref PubMed Scopus (341) Google Scholar). Heart lipids were extracted as described by Folch, Lees, and Sloane Stanley (25Folch 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). TG and cholesterol concentrations in hearts and plasma were measured using TG and cholesterol enzymatic assay kits (Infinity, Louisville, CO); FFAs were measured using NEFA C kits (Wako Chemicals, Richmond, VA). Tissue lipids were normalized by protein concentration. Cardiac and plasma SM levels were measured enzymatically as described previously (26Hojjati M.R. Jiang X.C. Rapid, specific, and sensitive measurements of plasma sphingomyelin and phosphatidylcholine.J. Lipid Res. 2006; 47: 673-676Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Cardiac ceramide and DAG levels were determined using the DAG kinase method (27Perry D.K. Bielawska A. Hannun Y.A. Quantitative determination of ceramide using diglyceride kinase.Methods Enzymol. 2000; 312: 22-31Crossref PubMed Google Scholar). Cardiac acyl CoAs were measured by liquid chromatography/tandem mass spectrometry using Perkin Elmer S200 HPLC (Perkin Elmer, Waltham, MA) and API 3000 (Applied Biosystems, Foster City, CA) and analyzed by Analyst 1.4.1 software (Applied Biosystems). Two-dimensional echocardiography was performed on conscious 14 to 16 week-old male (n = 10–12 per group) mice (Sonos 5500 system; Philips Medical Systems, Andover, MA) (28Takuma S. Suehiro K. Cardinale C. Hozumi T. Yano H. Shimizu J. Mullis-Jansson S. Sciacca R. Wang J. Burkhoff D. et al.Anesthetic inhibition in ischemic and nonischemic murine heart: comparison with conscious echocardiographic approach.Am. J. Physiol. Heart Circ. Physiol. 2001; 280: H2364-H2370Crossref PubMed Google Scholar). Echocardiographic images were recorded in a digital format. Images were then analyzed off-line by a single observer blinded to the murine genotype (29Wang C.Y. Mazer S.P. Minamoto K. Takuma S. Homma S. Yellin M. Chess L. Fard A. Kalled S.L. Oz M.C. et al.Suppression of murine cardiac allograft arteriopathy by long-term blockade of CD40-CD154 interactions.Circulation. 2002; 105: 1609-1614Crossref PubMed Scopus (47) Google Scholar). Quantitative real-time PCR was performed with SYBR Green PCR Core Reagents (Applied Biosystems). Incorporation of the SYBR green dye into the PCR products was monitored in real time with an Mx3000 sequence detection system (Stratagene, La Jolla, CA). Samples were normalized against β-actin. The sequences of the primers are provided in supplementary Table I. Isolated heart tissues were homogenized in PBS containing protease inhibitors and phosphatase inhibitors (Roche, Indianapolis, IN). Membrane and cytosolic fractions were separated by ultracentrifugation. Thirty micrograms from each fraction was applied to SDS-PAGE and transferred onto nitrocellulose membranes. GLUT4 and GLUT1 proteins in each fraction were detected by mouse-specific antibodies (Chemicon, Temecula, CA). Thirty micrograms of whole-tissue extracts was applied for Western blot analyses to examine phosphorylated (p) GSK-3β and pAKT, which were detected by mouse-specific antibodies (Cell Signaling, Danvers, MA). Basal glucose uptake was measured in hearts following an intravenous administration of 3 μCi of 2-deoxy-D-[1-14C]glucose (PerkinElmer Life Sciences). Blood was collected 30 s and 5, 30, and 60 min following injection. At 60 min, hearts were perfused with PBS, tissues were excised, and radioactive counts were measured. Hearts from 6 h-fasted mice were fixed in 10% formalin for 24 h and mounted on paraffin. Midventricular sections were stained with Schiff Reagent (Polyscientific, Bay Shore, NY) to identify glycogen in hearts [periodic acid-Schiff (PAS) staining] (30Yoshimura A. Toyoda Y. Murakami T. Yoshizato H. Ando Y. Fujitsuka N. Glycogen depletion in intrafusal fibres in rats during short-duration high-intensity treadmill running.Acta Physiol. Scand. 2005; 185: 41-50Crossref PubMed Scopus (5) Google Scholar). The specificity of glycogen staining was confirmed by treating sections with diastase to digest tissue glycogen, followed by regular PAS staining (30Yoshimura A. Toyoda Y. Murakami T. Yoshizato H. Ando Y. Fujitsuka N. Glycogen depletion in intrafusal fibres in rats during short-duration high-intensity treadmill running.Acta Physiol. Scand. 2005; 185: 41-50Crossref PubMed Scopus (5) Google Scholar). Sectioned heart tissues were stained for DNA fragmentation by a terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) staining protocol according to the manufacturer's instructions (R and D Systems, Minneapolis, MN). Hearts were hydrolyzed with 1 M NaOH at 65°C for 2 h, and glycogen was precipitated by 66% cold ethanol. Cardiac glycogen was digested by amyloglucosidase at 45°C for 2 h (31Ranalletta M. Jiang H. Li J. Tsao T.S. Stenbit A.E. Yokoyama M. Katz E.B. Charron M.J. Altered hepatic and muscle substrate utilization provoked by GLUT4 ablation.Diabetes. 2005; 54: 935-943Crossref PubMed Scopus (13) Google Scholar). The produced glucose was measured enzymatically by an AutoGlucose kit (Wako Chemicals). Cardiac metabolism data in isolated working hearts were analyzed by ANOVA, and significance was evaluated by the Fisher Least Protected Squares test. Differences among groups were determined using one-way ANOVA with post hoc Dunnett's t-test. A value of P < 0.05 was regarded as a significant difference. During myriocin treatment for 6 weeks, no significant changes in body weight, plasma glucose, cholesterol, TG, and FFA levels were observed (Table 1). Myriocin lowered plasma SM levels significantly in WT and LpLGPI mice.TABLE 1Body and heart weight, glucose, and plasma lipid measurements in WT and LpLGPI miceWTWT-myrLpLGPILpLGPI-myrBody wt. (g)29.8 ± 2.127.7 ± 1.529.5 ± 1.530.6 ± 3.5Heart wt./body wt. (%)0.58 ± 0.020.53 ± 0.060.64 ± 0.02aP < 0.05 (vs. WT).0.51 ± 0.02aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).Glucose (mg/dl)108 ± 3.9103 ± 6.0102 ± 7.7104 ± 6.4TG (mg/dl)70.5 ± 12.763.9 ± 4.878.0 ± 9.573.5 ± 2.4Cholesterol (mg/dl)69.1 ± 1.7574.5 ± 3.464.4 ± 4.774.1 ± 5.5FFA (mM)0.61 ± 0.120.44 ± 0.130.51 ± 0.080.43 ± 0.05SM (μg/ml)24.9 ± 2.312.1 ± 1.6aP < 0.05 (vs. WT).21.1 ± 5.09.1 ± 2.0aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).LpLGPI, glycosylphosphatidylinositol (GPI)-anchored human lipoprotein lipase; SM, sphingomyelin; myr, myriocin; TG, triglyceride; WT, wild type. Results are given as mean ± SEM, n = 8–10.a P < 0.05 (vs. WT).b P < 0.05 (vs. LpLGPI). Open table in a new tab LpLGPI, glycosylphosphatidylinositol (GPI)-anchored human lipoprotein lipase; SM, sphingomyelin; myr, myriocin; TG, triglyceride; WT, wild type. Results are given as mean ± SEM, n = 8–10. TG and cholesterol levels in mouse hearts were not changed by myriocin treatment or overexpression of LpL (Table 2). SM levels were 45% less in hearts of myriocin-treated LpLGPI than in untreated mice of the same genotype. In contrast, SM levels were not altered in WT mice (Table 2). Cardiac ceramide levels were increased approximately 45% in LpLGPI mouse hearts. Treatment with myriocin reduced ceramide to control levels (Table 2). Myriocin-treated WT mice did not show alteration of cardiac ceramide. This appeared to be due to upregulation of SPT subunits LCB1 and LCB2 as a compensatory mechanism to maintain basal sphingolipid pools (Table 3). DAG was also elevated in LpLGPI hearts, but myriocin had no effect on cardiac DAG levels (Table 2). Acyl CoAs were reduced in LpLGPI hearts, and myriocin treatment restored them to WT levels (Table 2). Thus, myriocin lowered cardiac SM and ceramide without changing TG, cholesterol, and DAG levels. Moreover, the improved hearts had a normalization of the reduced acyl CoA. We assumed that this was a secondary change due to reduced FA oxidation (see below).TABLE 2Cardiac lipid measurement in WT and LpLGPI miceWTWT-myrLpLGPILpLGPI-myrTG (μg/mg)16.8 ± 6.519.1 ± 9.018.1 ± 9.522.2 ± 8.0Cholesterol (μg/mg)4.2 ± 1.04.2 ± 0.185.0 ± 1.05.3 ± 0.7Fatty acyl CoA (pmol/mg)81.4 ± 6.2130.6 ± 4.8aP < 0.05 (vs. WT).41.4 ± 11.2aP < 0.05 (vs. WT).122.1 ± 4.8aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).SM (μg/mg)15.2 ± 2.514.6 ± 2.122.2 ± 1.9aP < 0.05 (vs. WT).12.2 ± 2.3aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).Ceramide (pmol/mg)210.5 ± 11.3216.3 ± 22.6305.9 ± 21.1aP < 0.05 (vs. WT).151.8 ± 9.9aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).DAG (pmol/mg)184.4 ± 4.9176.4 ± 8.5233.5 ± 5.6aP < 0.05 (vs. WT).228.0 ± 8.7aP < 0.05 (vs. WT).DAG, diacylglycerol. Results are given as mean ± SEM, n = 8–10.a P < 0.05 (vs. WT).b P < 0.05 (vs. LpLGPI). Open table in a new tab TABLE 3Expression of SPT subunits and heart failure markers in WT and LpLGPI mice4 month-oldGeneWTWT-myrLpLGPILpLGPI-myrLCB11.00 ± 0.142.19 ± 0.19aP < 0.05 (vs. WT).0.61 ± 0.05aP < 0.05 (vs. WT).1.19 ± 0.19bP < 0.05 (vs. LpLGPI).LCB21.00 ± 0.142.32 ± 0.38aP < 0.05 (vs. WT).0.87 ± 0.041.61 ± 0.22bP < 0.05 (vs. LpLGPI).ANF1.00 ± 0.090.81 ± 0.221.74 ± 0.20aP < 0.05 (vs. WT).0.47 ± 0.15aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).BNP1.00 ± 0.221.14 ± 0.172.27 ± 0.45aP < 0.05 (vs. WT).0.51 ± 0.12aP < 0.05 (vs. WT).,bP < 0.05 (vs. LpLGPI).SPT, serine palmitoyltransferase. Results are given as mean ± SEM, n = 8–10.a P < 0.05 (vs. WT).b P < 0.05 (vs. LpLGPI). Open table in a new tab DAG, diacylglycerol. Results are given as mean ± SEM, n = 8–10. SPT, serine palmitoyltransferase. Results are given as mean ± SEM, n = 8–10. LpLGPI hearts were hypertrophied with an increase in heart/body weight (Table 1; Fig. 1A). Myriocin treatment of these mice returned heart weights to WT levels. Echocardiography also revealed that hearts from chow-fed LpLGPI mice had left ventricular dilatation, increased left ventricular systolic diameter (LVD), and decreased fractional shortening, compared with hearts from chow-fed WT mice, as previously reported (19Yagyu H. Chen G. Yokoyama M. Hirata K. Augustus A. Kako Y. Seo T. Hu Y. Lutz E.P. Merkel M. et al.Lipoprotein lipase (LpL) on the surface of cardiomyocytes increases lipid uptake and produces a cardiomyopathy.J. Clin. Invest. 2003; 111: 419-426Crossref PubMed Scopus (295) Google Scholar). The LVDs of myriocin-treated LpLGPI mice were comparable to those of WT mice and were smaller than those of untreated LpLGPI mice (0.18 ± 0.014 cm vs. 0.24 ± 0.014 cm) (Fig. 1B). Reduced fractional shortening in LpLGPI mice was corrected by myriocin (46.1 ± 2.0% in myriocin-LpLGPI vs. 35.5 ± 2.0% in LpLGPI mice) (Fig. 1C). We assessed whether myriocin affected markers of cardiac failure. ANF and BNP gene expression was increased in LpLGPI hearts when compared with WT, and myriocin treatment reduced mRNA levels of these genes even further than those of WT (Table 3). Thus, treatment with myriocin that specifically reduced cardiac ceramide and had no impact on the levels of other major lipids improved cardiac function and reduced gene expression of cardiac failure markers. We assessed whether myriocin affected expression of metabolic genes in WT and LpLGPI hearts. GLUT4 was downregulated in LpLGPI hearts, and myriocin had no effect (Fig. 1D). GLUT1 expression was not altered by LpL overexpression or myriocin treatment (Fig. 1E). PDK4 mRNA was increased in hearts of LpLGPI mice (Fig. 1F). Increased PDK4 increases phosphorylation of pyruvate dehydrogenase and would be expected to reduce glucose oxidation rates in isolated hearts. Myriocin restored upregulation of PDK4 in LpLGPI hearts to WT levels. Expression levels of genes that regulate FA oxidation, such as PPAR-α, CPT-1, and ACO, were not changed in LpLGPI hearts by myriocin treatment (see supplementary Fig. I). Expression of fatty acid transporters such as CD36, acyl CoA synthase1 (ACS1), and fatty acid transport protein1 (FATP1) were downregulated in LpLGPI hearts (Fig. 1G, H, I). Myriocin restored them to the levels of WT. Cardiac metabolism was measured in hearts from 14 to 16 week-old male WT and LpLGPI mice. All hearts were prepared and perfused in the working mode, using previously described protocols (21Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Altered metabolism causes cardiac dysfunction in perfused hearts from diabetic (db/db) mice.Am. J. Physiol. Endocrinol. Metab. 2000; 279: E1104-E1113Crossref PubMed Google Scholar, 22Belke D.D. Larsen T.S. Gibbs E.M. Severson D.L. Glucose metabolism in perfused mouse hearts overexpressing human GLUT-4 glucose transporter.Am. J. Physiol. Endocrinol. Metab. 2001; 280: E420-E427Crossref PubMed Google Scholar, 23Belke D.D. Larsen T.S. Lopaschuk G.D. Severson D.L. Glucose and fatty acid metabolism in the isolated working mouse heart.Am. J. Physiol. 1999; 277: R1210-R1217Crossref PubMed Google Scholar, 24Mazumder P.K. O'Neill B.T. Roberts M.W. Buchanan J. Yun U.J. Cooksey R.C. Boudina S. Abel E.D. Impaired cardiac efficiency and increased fatty acid oxidation in insulin-resistant ob/ob mouse hearts.Diabetes. 2004; 53: 2366-2374Crossref PubMed Scopus (341) Google Scholar). Palmitate oxidation was increased by 27% and glucose oxidation was decreased by 26% in LpLGPI hearts relative to controls (Fig. 2A, B). Myriocin treatment reduced palmitate oxidation and increased glucose oxidation rates in LpLGPI hearts to the levels of WT (Fig. 2A, B). The average rates of glycolysis were not altered (Fig. 2C). Thus, hearts of LpLGPI mice develop altered substrate utilization with increased reliance on FAs, and myriocin altered substrate utilization by reducing FA oxidation and increasing glucose oxidation in LpLGPI hearts. In isolated working hearts, we did not detect any improvement in cardiac power following treatment of LpLGPI mice with myriocin (Fig. 2D). However, there was a significant reduction in MVO2 (Fig. 2E). This normalized cardiac efficiency (Fig. 2F), which was reduced in hearts from nontreated mice. These data suggest that myriocin enhanced myocardial energetics by maintaining cardiac performance at a lower oxygen cost. The gene expression data suggest that the increase in oxidation rates of exogenous palmitate in LpLGPI hearts might be the consequence of the PDK4-mediated reduction in pyruvate flux, which would reduce glucose oxidation. We examined whether in vivo glucose uptake was affected by myriocin using 2-deoxy-d-[1-3H]glucose (2-DG). There was no difference in plasma glucose clearance between WT and LpLGPI mice (Fig. 3A). In contrast to findings in isolated hearts (reduced glucose oxidation and normal rates of glycolysis), in vivo glucose uptake was increased in LpLGPI mouse hearts (Fig. 3B). Myriocin reduced in vivo glucose uptake in LpLGPI hearts to the levels of WT. The estimation of glycolytic rates in isolated hearts is based on the appearance of 3H, which is released as water at the final step in glycolysis, whereas 2-DG measures glucose transport and phosphorylation. If exogenous glucose uptake is increased but the glucose is not being oxidized, more glucose might be converted to glycogen; this would account for the differences between measurements obtained in vivo versus those obtained in isolated hearts. Histological analysis of heart tissue by PAS staining demonstrated that hearts of LpLGPI mice had greater glycogen stores than did hearts of myriocin-treated WT and LpLGPI mice (Fig. 3C). In contrast, hearts of LpLGPI mice treated with myriocin had a pattern of glycogen staining that was similar to that of WT mouse hearts (Fig. 3C). To confirm this, we biochemically measured cardiac glycogen. Glycogen content in hearts of LpLGPI mice was 49% greater than that in hearts of WT mice (Fig. 3D). Hearts of myriocin-treated LpLGPI mice had glycogen levels similar to those in hearts of WT mice (Fig. 3D). Thus, in hearts of LpLGPI mice, glucose carbons appear to preferentially accumulate as glycogen; myriocin treatment reversed this process. To assess whether the AKT/GSK-3β pathway was involved in hypertrophy and glycogen synthesis in hearts of LpLGPI mice, pAKT and its downstream target, pGSK-3β, were examined by Western blot. PAKT and pGSK-3β were elevated in hearts of LpLGPI mice, compared with hearts of WT mice (Fig. 3E–G). Myriocin treatment decreased pAKT and pGSK-3β levels in hearts of LpLGPI mice by 63% and 53%, respectively (Fig. 3E–G). GSK-3β blocks cardiac hypertrophy and inhibits glycogen synthesis. GSK-3β inactivation by phosphorylation would be predicted to promote cardiac hypertrophy and increase glycogen synthesis in the hearts of LpLGPI mice. Using recently developed human cardiomyocyte AC16 cells (20Davidson M.M. Nesti C. Palenzuela L. Walker W.F. Hernandez E. Protas L. Hirano M. Isaac N.D. Novel cell lines derived from adult human ventricular cardiomyocytes.J. Mol. Cell. Cardiol. 2005; 39: 133-147Abstract Full Text Full Text PDF PubMed Scopus (170) Google Scholar), we tried to reproduce conditions associated with lipotoxic cardiomyopathy and then examine whether ceramide alters
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