Diabetes-induced myelin abnormalities are associated with an altered lipid pattern: protective effects of LXR activation

• DOI: 10.1194/jlr.m021188
• Publication Date: 2011-12-09T01:24:42Z
• Authors: Gaia Cermenati; Federico Abbiati; Solei Cermenati; Elisabetta Brioschi; Alessandro Volont...; Guido Cavaletti; Enrique Saez; Emma De Fabiani; Maurizio Crestani; Luis M. Garcia-Se...; Roberto C. Melcangi; Donatella Caruso; Nico Mitro
• Keywords: Liver X receptor; Diabetic Neuropathy

Diabetic peripheral neuropathy (DPN) is characterized by myelin abnormalities; however, the molecular mechanisms underlying such deficits remain obscure. To uncover effects of diabetes on alterations, we have analyzed composition. In a streptozotocin-treated rat model diabetic neuropathy, analysis sciatic nerve lipids revealed that alters myelin's phospholipid, FA, and cholesterol content in pattern can modify membrane fluidity. Reduced expression relevant genes FA biosynthetic pathway decreased levels transcriptionally active form lipogenic factor sterol-regulatory element binding factor-1c (SREBF-1c) were found nerve. Expression major protein, protein zero (P0), was also suppressed diabetes. addition, confirmed induces abnormalities, primarily infoldings previously been associated with altered setting, synthetic activator nuclear receptor liver X (LXR) increased SREBF-1c function restored lipid species P0 to normal. 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Here, report display pattern, blunted pathway, chief (P0). Activation normalizes profile, restores reestablishes absolute P0, corrects nerves. improved tests. Our activation useful correct diabetes-associated All experiments conducted strictly following regulations European Community Directive 86/609/EEC (Official Journal L 358, 18/12/1986, pp. 0001–0028), local (e.g., Italian Legislative Decree n. 116, 27/01/1992) use laboratory animals. Animal protocols approved Institutional Review Committee. single intraperitoneal injection STZ, 65 mg/kg (Sigma-Aldrich; Milano, Italy) 0.09 M citrate buffer, pH 4.8, 2-month-old male Sprague-Dawley (Crl:CD BR, River; Lecco, Italy). Hyperglycemia verified after days, blood glucose concentrations >300 mg/dl included studies. Two months induction treated once week subcutaneous GW3965 (i.e., four month at 50 mg/kg) month. Nondiabetic vehicle. synthesized described receiving relative controls same protocol STZ-diabetic performed throughout 3 done temperature-controlled room adjacent animals' housing vital parameters monitored. internal standards (ISs) heneicosanoic (C21:0) 5α-cholestane, all analyses purchased Sigma-Aldrich. purified control, (31Melcangi Celotti Castano Martini Is 5 alpha-reductase-3 alpha-hydroxysteroid dehydrogenase complex young old rats?.Endocr. Regul. 1992; 119-125PubMed Internal added samples, extraction using Folch [chloroform-methanol (MeOH) 2:1, v/v]. Samples left overnight 4°C. Then organic residue divided two fractions: one free phospholipids (fraction A, total sample), B, 40% sample). Total obtained samples hydrolysis (32Taguchi Ishikawa Precise global identification phospholipid Orbitrap spectrometer automated search engine Lipid Search.J. Chromatogr. 1217: 4229-4239Crossref (155) Fraction B resuspended chloroform-MeOH 1:1, v/v. 1M HCl:MeOH (1:1, v/v) extract shaken h. chloroform-water added, lower collected, split, transferred into tubes dried under nitrogen flow. ml MeOH split 60/40 analysis, respectively. aliquot quantification suspended MeOH, concentrated, auto-sampler vials LC-MS/MS analysis. Quantitative basis calibration curves prepared standards. Electrospray ionization (ESI) linear ion trap-mass equipped Surveyor chromatography Pump Plus Autosampler Plus. LC mobile phases water MeOH/2-propanol (50:50, v/v). gradient (flow rate 0.2 ml/min) follows: T0: 100% T5: T5.1: 50% T25: T25.1: T40: Hypersil GOLDTM pentafluorophenyl column (100 mm × 2.1 mm, µm) maintained 40°C. volume 20 µl, injector needle washed MeOH-water 1:1 (v/v). Peaks off evaluated Dell workstation means software Excalibur, release 2.0 SR2. operated negative-ion mode ESI source sheath, auxiliary, sweeps gas, MS/MS helium collision gas. energy set 35% molecules. Principal component Unscrambler, version 10.1 (Camo Software; Oslo, Norway). Fractions quantitative cholesterol, first derivatized mixture bis-trimethylsilyltrifluoroacetamide-pyridine (4:1 30 min 60°C, then injected gas chromatograph-mass (GC-MS, Varian Saturn 2100). MS electron impact mode. GC-MS μl sample splitless (inlet kept 270°C flow 1.0 initial 180°C. oven 180°C min, ramped 50°C/min 240°C, 5°C/min 300°C 6 min. ions used m/z 368 357 IS. selection selective monitoring based spectra pure standards, freshly fixed concentration IS, different range 0 10 μg/μl. amount cholesteryl esters calculated subtracting cholesterol. Cholesteryl verify quality purity. accomplished either positive negative modes. infused acquisition spectra. Changes between detected families percent normalized analyzed. RNA Nucleospin® II kit (Macherey-Nagel; TaqMan CFX384 RT-qPCR iScriptTM one-step RT-PCR probes (Bio-Rad; arrayed 384-well format triplicate multiplexed reactions target gene 36B4 reference gene. Probe primer sequences Eurofins MWG-Operon (Milano, available request. constructs flag tagged mature forms SREBF-1a, SREBF-2 gift Dr. Osborne (Addgene plasmid #26801, #26802, #26807, respectively; Addgene, Cambridge, MA). Five micrograms each construct μg pcDNA3 (the backbone SREBFs) transfected individually Hek293T FuGENE (Promega; Italia) according manufacturing instructions 6-well tissue culture plates. Six hours transfection, cultured DMEM 10% FBS, 48 h later, extracts modified RIPA buffer [50 mM Tris-HCl (pH 7.4), 1% Nonidet P-40, 0.25% sodium deoxycholate, 150 NaCl, EDTA, protease inhibitor (Roche; Monza, Italy)]. Western blotting mouse monoclonal anti-SREBF-1 (Santa Cruz Biotechnology; dilution 1:200, polyclonal anti-Flag 1:1,000 (Sigma-Aldrich). Sciatic three per group lysed buffer. Biotechnology) rabbit anti-P0 (Sigma-Genosys; 1:500, anti-peripheral 22 (PMP22) (Inbios; Napoli, 1:5,000. loading probed anti-β-actin (Sigma-Aldrich) 1:1,000. secondary antibodies Quantification Quantity One (Bio-Rad). Analyses repeated least times sets analyze incubated slides 1:20 Slides goat anti-mouse Alexa Fluor 488 1:500 (Invitrogen, Italy), staining completed Hoechst 33258 (Invitrogen). Image Leica TCS SP2 AOBS Spectral confocal scanner mounted DM IRE2 inverted fluorescent microscope LCSTM software. 63× oil objective 2× digital zoom observations. compare conditions, fluorescence acquisitions always hardware settings (laser intensity, sampling, rate, pinhole, photomultiplier settings). localization assessed maximum projections (eight pictures stack). Regions interest drawn outline precisely nuclei within nuclei. quantity mean amplitude intensity (arbitrary unit). Fluorescence measured off-line, stack profile module image processing (Leica software; Nuclei fields slide preparations condition. For colocalization SREBF-1c-containing nuclei, counted preparations. removed, cut segments (1–2 length) immersion paraformaldehyde glutaraldehyde 0.1 phosphate 7.4, 24 Tissue postfixed buffered OsO4, dehydrated, embedded araldite. Nerves sectioned transversely Reichert Ultra-cut microtome. Semi-thin sections (0.5 stained toluidine blue light microscopy. Thin examined JEOL 1200 EXII microscope. morphometric fibers, areas semi-thin sections, covering 25% cross-sectional chosen systematic random sampling squares (33Mayhew T.M. Sampling schemes estimating fibre size. Methods trunks mixed fascicularity.J. Anat. 1984; 139: 45-58PubMed 34Mayhew I unifascicular trunks.J. 59-66PubMed size axons, g ratio, percentage normal irregular shapes, alteration compaction, ratio quotient axon (35Arbuthnott E.R. Boyd I.A. Kalu K.U. Ultrastructural dimensions fibres cat relation velocity.J. 1980; 308: 125-157Crossref (141) 36Arbuthnott Ballard K.J. non-circularity cat.J. 99-123Crossref (57) minimum 100 eight group. Statistical via one-way ANOVA followed Tukey-Kramer posttest. statistical GraphPad PRISM (San Diego, CA). adult injection. explore DPN, whether (through induce th