Secretion of fatty acid binding protein aP2 from adipocytes through a nonclassical pathway in response to adipocyte lipase activity

• DOI: 10.1194/jlr.m055798
• Publication Date: 2014-12-23T05:12:45Z
• Authors: Meric Erikci Ertunc; Jørgen Sikkeland; Federico Fenaroli; Gareth Griffiths; Mathew P. Daniels; Haiming Cao; Fahri Saatcioglu; Gökhan S. Hotamis...
• Keywords: Adipose triglyceride lipase; Hormone-sensitive lipase; Perilipin

Adipocyte fatty acid binding protein 4, aP2, contributes to the pathogenesis of several common diseases including type 2 diabetes, atherosclerosis, liver disease, asthma, and cancer. Although biological functions aP2 have classically been attributed its intracellular action, recent studies demonstrated that acts as an adipokine regulate systemic metabolism. However, mechanism regulation secretion remain unknown. Here, we demonstrate a specific role for lipase activity in from adipocytes vitro ex vivo. Our results show chemical inhibition activity, genetic deficiency adipose triglyceride and, lesser extent, hormone-sensitive blocked adipocytes. Increased lipolysis lipid availability also contributed release determined perilipin1-deficient tissue explants vivo upon treatment with lipids vitro. In addition, identify nonclassical route exosome-like vesicles is recruited this pathway stimulation lipolysis. 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Sel. 349-356Crossref (938) nonclassically protein. While consistent earlier resistant inhibitors brefeldin monensin Scholar), identity involved study, utilized approaches highly dependent lipases driven elevated lipids. Furthermore, found leads recruitment multivesicular bodies (MVBs), compartment, externalization vesicles. findings concept responds controlled indicate carry out, least part, functions. Plin1−/− obtained Jackson Laboratories (Bar Harbor, ME; stock number: 021887). Atglflox/flox (C57/BL6 background) kind gift Dr. Erin Kershaw (University Pittsburgh, PA), Hslflox/flox Maglflox/flox (mixed Rudolf Zechner Graz, Austria). Adiponectin-Cre (Adipoq-Cre) Evan Rosen (Beth Israel Deaconess Medical Center, Harvard School) backcrossed onto C57Bl/6J background. order obtain tissue-specific lipase-deficient mice, homozygous floxed crossed Adipoq-Cre mice. Cre-positive progeny then flox/flox flox/flox;Cre use experiments. prepared 10- 16-week-old Intralipid infusion experiments performed 24-week-old male All maintained 12 h light dark cycle. regular chow (RD, PicoLab 5058 Lab Diet, 9% fat). Area Standing Committee Animals approved all studies. Conditioned media (CM), cell lysates (CLs), (TLs), run 15% SDS-PAGE gels. For immunoblotting, rabbit anti-aP2 was produced in-house recombinant, full-length aP2. Other antibodies following commercial sources: β-tubulin (Santa Cruz, sc-9104), (for confocal electron microscopy, Signaling, 3544), anti-adipose (ATGL) (Cell Signaling Technology, 2138), anti-hormone-sensitive (HSL) 4107S), anti-perilipin sc-47322), anti-milk globule-EGF Factor 8 (MFG-E8; Santa sc-33546), anti-protein disulfide isomerase (PDI; Stressgen, SPA-901), anti-cluster 63 (CD63; sc-15363), anti-Programmed death 6 interacting (ALIX; BioLegend, 634501), anti-tumor susceptibility 101 (TSG101; Abcam, ab83). FLAG-tagged immunoprecipitated conditioned anti-FLAG M2 affinity agarose gel (Sigma) overnight 4°C. Proteins eluted 2× SDS loading buffer analyzed Western blots. used 1:1,000 dilution, detected BM chemiluminescence blotting substrate (Roche) SuperSignal West Femto (Pierce). FLAG blots quantified ImageJ software (The National Institutes Health, Bethesda, MD). previously described (33Nakamura Cao Sonenberg Double-stranded RNA-dependent kinase links pathogen sensing homeostasis.Cell. 140: 338-348Abstract (385) Briefly, WT fasted before infused 5 ml/kg/h (Baxter Healthcare Corporation) h. Blood collected after infusion. Plasma separated microcentrifugation blood 13,000 rpm 30 min. ELISA system (Biovendor Inc.). 3T3-L1 derived WT, aP2-deficient, (aP2−/−; mal1−/−)-deficient preadipocytes DMEM 10% bovine calf serum. differentiation, seeded, medium changed days (day −2). On day 0, induced Cosmic Calf (CCS) 500 µM 3-isobutyl-1-methylxanthine (IBMX), µg/ml insulin, 10 dexamethasone, rosiglitazone. switched CCS thereafter, supplemented only insulin. Lipolytic without done 8–12 induction. induction treated 1 mM IBMX 20 forskolin (FSK) h, refreshed another hour. Medium microcentrifuged 5,000 Supernatants further analysis. involving inhibitors, differentiated pretreated H89 (50 µM), Atglistatin (kind Zechner, University Austria) (10 76-0079 Peter Kurtzhals Christian Fledelius, Novo Nordisk, Denmark) (2.5 CAY10499 (0.1 μM), DMSO Then cotreated explained above. experiments, stocks free acids acid-free, low-endotoxin BSA diluted CCS. incubated vehicle (500 µM) 24 processed extracellular vesicle (EV) isolation stimulation, indicated stimulants 3 below isolate adipose-specific models. Plin1+/− intercrossed littermates. Perigonadal removed preparation explants. samples washed PBS containing consecutively minced roughly mm-size pieces scissors. Explants same medium. lipase-deficiency models, IBMX, FSK, dibutyryl-cAMP (N6,2′-O-dibutyryladenosine 3′,5′-cyclic monophosphate sodium salt), isoproterenol, CL 316,243, culture evaluated protocol perilipin1-deficiency model, 16 assess extent under basal conditions. Glycerol glycerol determination (Sigma-Aldrich), NEFA HR Series NEFA-HR (2Scherer (Wako Diagnostics) normalized total lysates. cloned pcDNA3 plasmid (Invitrogen). mutant (LBM) version GeneTailorTM Site-directed mutagenesis endogenous (aP2−/−) electroporated Line L Nucleofector (Lonza) AMAXA electroporator. next day, 13 differentiation. green fluorescent (GFP)-aP2 cloning lentiviral vector pRRLCMVGFP. infected lentivirus microscopy 7 express FLAG-tagged-GFP-aP2. 8, kept F12 They water isoproterenol μM) Cells fixed 4% formaldehyde 15 min stained LipidTOX Red 4,6-diamidino-2-phenylindole (DAPI) visualizing droplets nuclei, respectively. (4% w/v 200 HEPES pH 7.4 buffer) room temperature. two times infiltrated solution 2.3 sucrose 48 Subsequently, 0.5% centrifuged pellet. This embedded gelatin cryosectioned (34Griffiths Simons Warren Tokuyasu Immunoelectron thin, frozen sections: application transport Semliki Forest virus spike glycoproteins.Methods Enzymol. 1983; 466-485Crossref (161) Sections picked up 1:1 2% methyl cellulose sucrose. labeled GFP followed gold nm) final visualization transmission microscopy. Isolated adsorbed nickel mesh grids had coated formvar film subjected glow-discharge increase hydrophilicity. suspensions 4°C 0.1 phosphate buffer, 7.3. sequential washes incubations immunolabeling temperature: four PBS; permeabilization blocking 0.075% saponin PBS, normal goat serum, 1% min; (1:100) (PBS-BSA) h; PBS-BSA 1.4 nm gold-conjugated Fab′ anti-rabbit IgG (Nanoprobes, 1:100) three refixation glutaraldehyde 7.3 After washing water, immunogold-labeled silver-enhanced HQ silver enhancement kit (Nanoprobes) again positive staining uranyl acetate. Samples stabilized carbon coating prior imaging JEM 1200EX microscope (JEOL USA) equipped AMT XR-60 digital camera (Advanced Microscopy Techniques Corp.). Differentiated induce above, time points indicated. Microsomes fractionated (35Cox B. Emili Tissue subcellular fractionation extraction mass-spectrometry-based proteomics.Nat. Protoc. 1: 1872-1878Crossref (259) homogenized Teflon pestle, nuclei cleared 800 g, mitochondria 6,000 g. supernatant ul­tracentrifuged 100,000 g pellet microsomes. resuspended lysis blot. EV-depleted conditioning ultracentrifugation 20% 18 28,500 (∼100,000 g) SW32 rotor (Beckman Coulter). Media filtered (0.2 µm) DMEM. vesicle-depleted conditioning. Processing point min, twice, 2,000 eliminate floating cells. 0.45 µm pore filters ultracentrifuged 9,000 clear debris. Supernatant 90 repell