Autophagy regulates sphingolipid levels in the liver

• DOI: 10.1194/jlr.m051862
• Publication Date: 2014-10-21T01:43:54Z
• Authors: Aikaterini Alexaki; Sita D. Gupta; Saurav Majumder; Mari Kono; Galina Tuymetova; Jeffrey M. Harmon; Teresa M. Dunn; Richard L. Proia
• Keywords: Sphingolipid; Autophagosome

Sphingolipid levels are tightly regulated to maintain cellular homeostasis. During pathologic conditions such as in aging, inflammation, and metabolic neurodegenerative diseases, of some sphingolipids, including the bioactive metabolite ceramide, elevated. metabolism has been linked autophagy, a critical catabolic process both normal cell function disease; however, vivo relevance interaction is not well-understood. Here, we show that blocking autophagy liver by deletion Atg7 gene, which essential for autophagosome formation, causes an increase sphingolipid metabolites ceramide. We also overexpression serine palmitoyltransferase elevate de novo biosynthesis induces liver. The results reveal limits excessive ceramide induced elevation synthesis Dysfunctional may be underlying mechanism causing elevations contribute pathogenesis diseases. Sphingolipids structurally functionally diverse family lipids serve membrane components signaling molecules (1Breslow D.K. Weissman J.S. 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PCR mouse-tail DNA genotype GFP-LC3, Atg7lox, loci; primers all PCRs 44Mizushima Methods monitoring GFP-LC3 mice.Methods Enzymol. 452: 13-23Crossref (136) Mice infected tail-vein injection × 108 plaque forming units (PFU) fSPT-adenovirus (or Mut-fSPT-adenovirus) low-dose model 109 PFU same viruses high-dose model. fed regular chow diet [or high-fat (60% kcal fat, Research Diets, Inc.) indicated]. animal procedures approved National Institute Diabetes Digestive Kidney Diseases Animal Care Use Committee accordance Institutes Health guidelines. Microsomes prepared (45Han Gable Yan Natarajan Krishnamurthy Borovitskaya topology Lcb1p subunit yeast palmitoyltransferase.J. 53707-53716Abstract (49) Microsomal assayed measuring acyl-CoA-dependent [3H]serine long-chain bases (46Han Moitra Eichler Brown R.H. Identification small subunits mammalian confer distinct acyl-CoA substrate specificities.Proc. 106: 8186-8191Crossref (175) Microsome preparations livers adenovirus-injected immunoblotting anti-sptlc1 (BD Biosciences) antibody. Total homogenate experiments. homogenized RIPA buffer (Boston BioProducts) protease inhibitors (Thermo Scientific), crude centrifuged 8,000 g, resulting supernatant stored −80°C. PVDF anti-LC3B (Sigma-Aldrich); nitrocellulose cases. Chemiluminescence detection analyzing samples SPT-adenovirus-injected mice; Atg7indKO, Atg7LKO, LI-COR Odyssey used. plasma homogenates measured high-performance liquid chromatography-tandem mass spectrometry Lipidomics Core Medical University South Carolina Thermo Finnigan TSQ 7000 triple quadrupole spectrometer, operating reaction monitoring-positive ionization mode (47Pettus B.J. Bielawski Porcelli Reames D.L. Johnson K.R. Morrow Chalfant 1/sphingosine-1-phosphate mediates COX-2 PGE2 production TNF-alpha.FASEB 17: 1411-1421Crossref (294) PBS BCA assay Scientific). Lipids extracted chloroform/methanol (2:1), dried overnight, resuspended isopropanol. Triglyceride reagent (Pointe Scientific, Inc.). perfused 4% paraformaldehyde. Fixed embedded OCT medium. Oil Red O (ORO) staining Histoserv, Inc. Sections examined Leica DMLB microscope. For confocal microscopy, 8 μm-thick sections cut fixed again paraformaldehyde counterstained 4',6-diamidino-2-phenylindole (DAPI). LSM Live Duoscan (Zeiss) microscope analyzed Zen 2009 software. electron 1% glutaraldehyde/4% 4°C 24 h. Further processing JFE Enterprises. (Philips EM 410). RNA purified RNeasy mini kit (Qiagen). μg) digested DNase I reverse-transcribed SuperScript First-Strand (Invitrogen) following manufacturer's instructions. Expression mRNA determined predesigned assay-on-demand probes (Applied Biosystems) ABI Prism 7700 sequence Biosystems). Unpaired Student's t-tests compare between different groups. P < 0.05 considered statistically significant. sought inducible Atg7, formation. these autophagy-defective (Atg7-indKO, having Atglox/lox Mx-cre), deleted tissues adult stimulation recombinase administration quantified (Atg7-indKO) (Atg7lox/lox) mice. Mass dihydroceramide, sphingosine, dihydrosphingosine, S1P, dihydro-S1P. Compared showed significant total those individual species (Fig. 1A). Dihydrosphingosine, These indicate major limiting investigate SPT, enzyme initiates biosynthesis, increased, Sptlc1 Sptlc2. Immunoblot demonstrated substantial Sptlc2 compared 1B). enzymatic significantly 1C). 1D), indicating altered transcriptional regulation did activity. Interestingly, unlike calnexin, another resident ER, 1B), suggesting autophagy-specific effect toward SPT. confirm seen secondary extrahepatic conducted experiments above line solely liver-specific (Atg7LKO) driven albumen promoter. genotype, albumen-Cre recapitulated (supplementary Fig. I) levels. Collectively, block large impact positive regulator heterotrimer consisting two (SPTLC1 SPTLC2 its functional isoform SPTLC3) one (ssSPT isoforms b). facilitate contained SPTLC1, SPTLC2, ssSPTa single in-frame polypeptide (SPTLC2-ssSPTa-SPTLC1), utilized 2A). protein, had similar wild-type 48Harmon Bacikova Sengupta Somashekarappa Topologi­cal characterization ssSPTs, activating 10144-10153Abstract incorporated (fSPT-adenovirus). point inactivation (49Gable Monaghan Williams Mutations LCB1 LCB2 genes, corresponding hereditary sensory neuropathy mutations, dominantly inactivate 10194-10200Abstract (94) (Mut-fSPT-adenovirus). second no (null-adenovirus) Intravenous adenoviruses liver-directed evaluated doses [low dose: plaque-forming (PFU)/mouse; high PFU/mouse] Immunoblotting antibody SPTCL1, reacts endogenous Sptlc1. very highly relative highest dose 2B). Correspondingly, level 12-fold higher microsomes isolated fSPT-adenovirus-injected control-adenovirus-injected 2C). At low dose, readily detectable day progressively until 10; 14, substantially declined 2D). protein-expression data correlated 2E), 3-fold over 10 decline 14. dihydro-S1P fSPT-adenovirus. Most treatment; 2-fold 3.5-fold plasma, respectively 3A, B). observed dihydrosphingosine dihydroceramide plasma. sphingolipids generated during initial steps before concentrations consistent flux pathway. further down but treated fSPT-adenovirus, albeit lower extent dose. 10, approximately (19% 22%, respectively) 4A, Increases increases were, most part, IIA, Administration produced serum alanine aminotransferase aspartate treatment adenovirus, expressio