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Lactosylceramide contributes to mitochondrial dysfunction in diabetes

2016; Elsevier BV; Volume: 57; Issue: 4 Linguagem: Inglês

10.1194/jlr.m060061

1539-7262

Sergei A. Novgorodov, Christopher L. Riley, Jin Yu, Jarryd A. Keffler, Christopher J. Clarke, An O. Van Laer, Catalin F. Baicu, Michael R. Zile, Tatyana I. Gudz,

Lipid Membrane Structure and Behavior

Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes. Sphingolipids have been implicated as key mediators of cell-stress responses and effectors of mitochondrial function. To investigate potential mechanisms underlying mitochondrial dysfunction, an important contributor to diabetic cardiomyopathy, we examined alterations of cardiac sphingolipid metabolism in a mouse with streptozotocin-induced type 1 diabetes. Diabetes increased expression of desaturase 1, (dihydro)ceramide synthase (CerS)2, serine palmitoyl transferase 1, and the rate of ceramide formation by mitochondria-resident CerSs, indicating an activation of ceramide biosynthesis. However, the lack of an increase in mitochondrial ceramide suggests concomitant upregulation of ceramide-metabolizing pathways. Elevated levels of lactosylceramide, one of the initial products in the formation of glycosphingolipids were accompanied with decreased respiration and calcium retention capacity (CRC) in mitochondria from diabetic heart tissue. In baseline mitochondria, lactosylceramide potently suppressed state 3 respiration and decreased CRC, suggesting lactosylceramide as the primary sphingolipid responsible for mitochondrial defects in diabetic hearts. Moreover, knocking down the neutral ceramidase (NCDase) resulted in an increase in lactosylceramide level, suggesting a crosstalk between glucosylceramide synthase- and NCDase-mediated ceramide utilization pathways. These data suggest the glycosphingolipid pathway of ceramide metabolism as a promising target to correct mitochondrial abnormalities associated with type 1 diabetes. Compelling epidemiological and clinical data indicate that diabetes mellitus increases the risk for cardiac dysfunction and heart failure independently of other risk factors, such as coronary disease and hypertension. Lipotoxicity is an important contributor to cardiac dysfunction in both type 1 (1Basu R. Oudit G.Y. Wang X. Zhang L. Ussher J.R. Lopaschuk G.D. Kassiri Z. Type 1 diabetic cardiomyopathy in the Akita (Ins2WT/C96Y) mouse model is characterized by lipotoxicity and diastolic dysfunction with preserved systolic function.Am. J. Physiol. Heart Circ. 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Diabetic cardiomyopathy, causes and effects.Rev. Endocr. Metab. Disord. 2010; 11: 31-39Crossref PubMed Scopus (509) Google Scholar) diabetes, which are characterized by excessive accumulation of triacylglycerols, long-chain acyl-CoAs, diacylglycerols, and ceramides in myocardium. Correlation of the increased ceramide level with development of diabetic cardiomyopathy has attracted special attention in recent years because of the well-documented ability of this sphingolipid to regulate a number of cell processes, including cell proliferation, growth arrest, differentiation and apoptosis, and the mediation of responses to stress stimuli. Ceramide is a hub of sphingolipid metabolism (7Hannun Y.A. Obeid L.M. Principles of bioactive lipid signalling: lessons from sphingolipids.Nat. Rev. Mol. Cell Biol. 2008; 9: 139-150Crossref PubMed Scopus (2442) Google Scholar, 8Lahiri S. Futerman A.H. The metabolism and function of sphingolipids and glycosphingolipids.Cell. Mol. Life Sci. 2007; 64: 2270-2284Crossref PubMed Scopus (262) Google Scholar). The balance between ceramide-producing pathways and pathways that consume it dictates ceramide level in the cell. The de novo ceramide biosynthesis pathway, one of the major ceramide producing pathways, localizes in the endoplasmic reticulum (ER) and starts with the condensation of serine and palmitoyl-CoA producing 3-keto-sphingonine, which, in turn, is rapidly converted to dihydrosphingosine. Subsequent acylation of dihydrosphingosine by a set of (dihydro)ceramide synthases (CerSs) gives rise to dihydroceramide. Finally, the removal of two hydrogens from the fatty acid chain of dihydroceramide by desaturase leads to the formation of ceramide. Other possible contributors to elevated ceramide level are: hydrolysis of SM catalyzed by SMases, acylation of sphingosine in the salvage pathway, dephosphorylation of ceramide-1-phosphate, and catabolism of glycosphingolipids. Ceramide levels are tightly controlled in the cells via rapid transformation into less harmful sphingolipids. The main pathway of ceramide utilization is its conversion to sphingosine and FFA catalyzed by ceramidases, which reside in mitochondria, plasma membrane, lysosomes, and Golgi compartments (neutral, acid, and alkaline ceramidases). Ceramide can also be channeled to the formation of complex glycosphingolipids. The first step in this pathway is catalyzed by glucosylceramide synthase with the formation of glucosylceramide. Subsequent formation of lactosylceramide and further gangliosides gives rise to a wide spectrum of biologically active molecules. Conversion to SM (SM synthase) and ceramide-1-phosphate (ceramide kinase) are other alternatives for ceramide utilization. Mitochondria emerged as a novel specialized compartment of sphingolipid metabolism with their own subset of sphingolipid-generating and -degrading enzymes. Neutral ceramidase (NCDase) (9Novgorodov S.A. Wu B.X. 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The goal of this investigation was to determine the effect of type 1 diabetes, induced by streptozotocin (STZ), on cardiac mitochondrial sphingolipid makeup and identify the sphingolipids contributing to mitochondrial dysfunction. We evaluated two distinct mitochondrial subpopulations: subsarcolemmal mitochondria (SSM) and interfibrillar mitochondria (IFM), which differ with respect to their respiratory capacity, sensitivity to Ca2+, and response to type 1 diabetic insult (41Palmer J.W. Tandler B. Hoppel C.L. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle.J. Biol. Chem. 1977; 252: 8731-8739Abstract Full Text PDF PubMed Google Scholar, 42Hollander J.M. Thapa D. Shepherd D.L. Physiological and structural differences in spatially distinct subpopulations of cardiac mitochondria: influence of cardiac pathologies.Am. J. Physiol. Heart Circ. Physiol. 2014; 307: H1-H14Crossref PubMed Scopus (101) Google Scholar). There were no substantial increases in ceramide level in both subpopulations in diabetic heart tissue, despite an upregulation of CerS2, serine palmitoyl transferase (SPT) 1, and desaturase 1 expression, indicative of activation of the de novo ceramide biosynthesis pathway. This suggests concert activation of ceramide-producing and ceramide-utilizing reactions. Indeed, NCDase-deficient mice display increased ceramide levels after induction of diabetes, suggesting NCDase-mediated ceramide hydrolysis as an important checkpoint in the control of mitochondrial ceramide. An activation of ceramide-consuming pathways gains additional support due to the substantial rise in lactosylceramide upon induction of diabetes in WT mice, which was further enhanced in NCDase-null mice, especially in IFM. Evaluation of the effects of exogenous lactosylceramide on baseline IFM revealed suppression of the respiratory chain and decreased calcium retention capacity (CRC). These studies suggest that type 1 diabetes does not induce ceramide accumulation in heart mitochondria, but rather increases ceramide flux, and that, not ceramide, but ceramide-derived glycosphingolipid lactosylceramide mediates the mitochondrial dysfunction. Male C57BL/6 mice were from The Jackson Laboratory (Sacramento, CA). NCDase KO mice were generated in the laboratory of Dr. Richard Proia (43Kono M. Dreier J.L. Ellis J.M. Allende M.L. Kalkofen D.N. Sanders K.M. Bielawski J. Bielawska A. Hannun Y.A. Proia R.L. Neutral ceramidase encoded by the Asah2 gene is essential for the intestinal degradation of sphingolipids.J. Biol. Chem. 2006; 281: 7324-7331Abstract Full Text Full Text PDF PubMed Scopus (121) Google Scholar) (National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health) and transferred to the animal facility of the Medical University of South Carolina. The mice were C57BL/6 background and were backcrossed for 10 additional generations with the same background at the Medical University of South Carolina. Experimental protocols were reviewed and approved by the Insti­tutional Animal Care and Use Committee of the Medical University of South Carolina (Charleston, SC) and followed the National Institutes of Health guidelines for experimental animal use. Diabetes was induced in 8- to 9-week-old mice following the Low-Dose Streptozotocin Induction Protocol (Mouse) of the Diabetic Complications Consortium. Animals were fasted for 4 h prior to STZ injection. Injections (ip) were performed at 50 mg/kg body weight of STZ dissolved in sodium citrate buffer (pH 4.5) for five consecutive days. Plasma glucose levels were monitored with TRUEread blood glucose monitor (Nipro-Diagnostics, Fort Lauderdale, FL) and animals with glucose levels >300 mg/dl were considered diabetic. Control animals were given injections of citrate buffer. Alternatively, animals with diabetes induced by the similar protocol were obtained from the Jackson Laboratory. Five weeks postinjection animals were used for experimentation. TPCK-treated trypsin and soybean trypsin inhibitor were from Worthington Biochemical Corporation (Lakewood, NJ). According to manufacturer's specification, 1 mg inhibitor suppresses activity of 2.06 mg trypsin. The 17C-sphingosine, palmitoyl-CoA, arachidoyl-CoA, C16:0-lactosylceramide, and C16:0-glucosylceramide were from Avanti Polar Lipids (Alabaster, AL). All other chemicals were purchased from Sigma-Aldrich (St. Louis, MO). Antibodies against mitochondrial marker, voltage dependent anion channel (VDAC) (D73D12), and syntaxin 6 (Golgi marker) were supplied by Cell Signaling Technology (Danvers, MA). The rabbit polyclonal anti-LAMP-2 (lysosomal marker), mouse monoclonal anti-α1 subunit of the Na+/K+ ATPase (plasma membrane marker), and the rabbit polyclonal anti-calnexin (ER marker) antibodies were purchased from Abcam (Cambridge, MA). Rabbit polyclonal anti-SPTLC1 antibody was purchased from Abcam. Goat polyclonal anti-SPTLC2 (C20), rabbit polyclonal anti-SPTLC3 (D-13), rabbit polyclonal anti-UGCG (H-300), and rabbit polyclonal anti-CerS2 antibodies were purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Rabbit polyclonal anti-NCDase antibody was raised by Bethyl Laboratories (Montgomery, TX) (10Novgorodov S.A. Riley C.L. Yu J. Borg K.T. Hannun Y.A. Proia R.L. Kindy M.S. Gudz T.I. Essential roles of neutral ceramidase and sphingosine in mitochondrial dysfunction due to traumatic brain injury.J. Biol. Chem. 2014; 289: 13142-13154Abstract Full Text Full Text PDF PubMed Scopus (33) Google Scholar). Mouse monoclonal anti-β-actin antibody (AC-74) was purchased from Sigma-Aldrich. Secondary horseradish peroxidase-conjugated antibodies were supplied by Jackson ImmunoResearch Laboratories Inc. (West Grove, PA). Mice underwent echocardiography to examine in vivo changes in cardiac function upon induction of diabetes using a 40 MHz mechanical scanning transducer (707B) and a Vevo 770 echocardiograph (VisualSonics, Toronto, Canada), as previously described (44Zile M.R. Baicu C.F. Stroud R.E. Van Laer A. Arroyo J. Mukherjee R. Jones J.A. Spinale F.G. Pressure overload-dependent membrane type 1-matrix metalloproteinase induction: relationship to LV remodeling and fibrosis.Am. J. Physiol. Heart Circ. Physiol. 2012; 302: H1429-H1437Crossref PubMed Scopus (32) Google Scholar). Left ventricular (LV) weight (LVW), LV ejection fraction (EF), stroke volume (SV), and left atrial (LA) dimension were measured using the American Society of Echocardiography criteria (45Lang R.M. Bierig M. Devereux R.B. Flachskampf F.A. Foster E. Pellikka P.A. Picard M.H. Roman M.J. Seward J. Shanewise et al.Recommendations for chamber quantification: a report from the American Society of Echocardiography's Guidelines and Standards Committee and the Chamber Quantification Writing Group, developed in conjunction with the European Association of Echocardiography, a branch of the European Society of Cardiology.J. Am. Soc. Echocardiogr. 2005; 18: 1440-1463Abstract Full Text Full Text PDF PubMed Scopus (9444) Google Scholar). LVW was normalized to body weight. SSM and IFM were isolated according to Palmer, Tandler, and Hoppel (41Palmer J.W. Tandler B. Hoppel C.L. Biochemical properties of subsarcolemmal and interfibrillar mitochondria isolated from rat cardiac muscle.J. Biol. Chem. 1977; 252: 8731-8739Abstract Full Text PDF PubMed Google Scholar) and King et al. (34King K.L. Young M.E. Kerner J. Huang H. O'Shea K.M. Alexson S.E. Hoppel C.L. Stanley W.C. Diabetes or peroxisome proliferator-activated receptor alpha agonist increases mitochondrial thioesterase I activity in heart.J. Lipid Res. 2007; 48: 1511-1517Abstract Full Text Full Text PDF PubMed Scopus (38) Google Scholar) with some modifications. Hearts from eight to ten mice were minced and washed in isolation buffer (IB) consisting of 100 mM KCl, 50 mM MOPS, 1 mM EGTA, 5 mM MgSO4, and 1 mM ATP (pH 7.4 adjusted by KOH). Minced tissue was diluted 1:10 (w/v) in IB and homogenized with a Polytron tissue processor (Brinkman Instruments, Westbury, NY) for 3 s at a rheostat setting of 6.5, and further homogenized with a motorized Potter-Elvejhem homogenizer by five strokes at 800 rpm. Homogenate was centrifuged at 600 g for 10 min. Supernatant was saved and the pellet was resuspended in IB. After repeated centrifugation at 600 g for 10 min, the supernatants were combined and the pellet was saved for isolation of IFM. SSM were pelleted from supernatant by centrifugation at 3,100 g for 10 min. To isolate IFM, pellet from the last centrifugation at 600 g was suspended in IB containing trypsin (5 mg/g wet weight) for 10 min at 4°C. Digestion was terminated by addition of an equal volume of IB containing soybean trypsin inhibitor (5 mg/g wet weight) and the suspension was centrifuged at 7,700 g for 10 min. Supernatant was discarded and myofibril pellet was resuspended in IB and spun down at 600 g for 10 min. Supernatant was saved and the procedure was repeated one more time. IFM were obtained from combined supernatants by centrifugation at 3,100 g for 10 min. To increase purification of SSM and IFM, pellets were resuspended in IB and spun down at 3,100 g for 10 min. This step was repeated one more time, however, mitochondria were resuspended in a buffer containing 100 mM KCl, 50 mM MOPS, and 0.5 mM EGTA (pH 7.4 adjusted by KOH). After final centrifugation, mitochondria were resuspended in the above mentioned buffer. Mitochondria had negligible contamination with the major cellular membrane compartments, as assessed by Western blot using antibodies against plasma membrane marker protein (Na+,K+-ATPase), lysosomes (LAMP-2), ER (calnexin), and Golgi marker (syntaxin 6) (Fig. 1). As expected, the outer mitochondrial membrane marker VDAC was enriched in the mitochondrial fractions as compared with homogenate. Mitochondrial protein concentration was quantified with a bicinchoninic acid assay kit (Pierce, Rockford, IL) using BSA as a standard. Inner membrane permeabilization was assayed by measurements of the decrease in absorbance of mitochondrial suspension, indicative of mitochondrial swelling, at 520 nm using a Brinkmann PC 910 probe colorimeter and a fiber optic probe, as we described (20Novgorodov S.A. Gudz T.I. Obeid L.M. Long-chain ceramide is a potent inhibitor of the mitochondrial permeability transition pore.J. Biol. Chem. 2008; 283: 24707-24717Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar). The CRC of mitochondria was monitored using a Ca2+-selective electrode (ThermoScientific/Orion, Rockford, IL) in a medium containing 250 mM sucrose, 10 mM HEPES, and 2 mM KH2PO4 (pH 7.4) (adjusted with Tris-base) at 25°C, as previously described (20Novgorodov S.A. Gudz T.I. Obeid L.M. Long-chain ceramide is a potent inhibitor of the mitochondrial permeability transition pore.J. Biol. Chem. 2008; 283: 24707-24717Abstract Full Text Full Text PDF PubMed Scopus (35) Google Scholar, 46Soriano M.E. Nicolosi L. Bernardi P. Desensitization of the permeability transition pore by cyclosporin a prevents activation of the mitochondrial apoptotic pathway and liver damage by tumor necrosis factor-alpha.J. Biol. Chem. 2004; 279: 36803-36808Abstract Full Text Full Text PDF PubMed Scopus (64) Google Sc