Loss of perilipin 2 in cultured myotubes enhances lipolysis and redirects the metabolic energy balance from glucose oxidation towards fatty acid oxidation

• DOI: 10.1194/jlr.m079764
• Publication Date: 2017-08-20T00:10:16Z
• Authors: Yuan Z. Feng; Jenny Lund; Yuchuan Li; Irlin K. Knabenes; Siril S. Bakke; Eili T. Kase; Yun K. Lee; Alan R. Kimmel; G. Hege Thoresen; Arild Christian R...; Knut Tomas Dalen
• Keywords: Perilipin

Lipid droplet (LD) coating proteins are essential for the formation and stability of intracellular LDs. Plin2 is an abundant LD protein in skeletal muscle, but its importance muscle function unclear. We show that myotubes established from Plin2−/− mice contain reduced content LDs accumulate less oleic acid (OA) triacylglycerol (TAG) due to elevated hydrolysis comparison with Plin2+/+ myotubes. The ability store TAG accompanied by a shift energy metabolism. characterized increased oxidation OA, lower glycogen synthesis, glucose myotubes, perhaps reflecting competition between FAs as part Randle cycle. In accord these metabolic changes, have expression Ppara Ppargc1a, transcription factors stimulate genes important FA oxidation, whereas involved uptake suppressed. Loss had no impact on insulin-stimulated Akt phosphorylation. Our results suggest protecting pool avoid uncontrolled stored balance Energy drive contraction fibers obtained primarily or FAs. 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DMEM (DMEM-Glutamax™, 5.5 mM glucose) sodium pyruvate, without phenol red, Ham's F-10-Glutamax™ nutrient mixture (5.5 glucose), horse serum, heat-inactivated FBS, basic fibroblast growth factor, collagenase II, penicillin-streptomycin, amphotericin B, Bodipy 493/503, Hoechst 33258, 33342, ABI 6100 Nucleic Acid Prep-Station solutions, Superscript III RT were Thermo Fisher Scientific (Waltham, MA). Matrigel was purchased BD Biosciences (Bedford, Insulin (Actrapid®) Novo Nordisk (Bagsvaerd, Denmark). BSA (essentially free), L-carnitine, Dulbecco's PBS (DPBS, Mg2+ Ca2+), (OA; 18:1, n-9), Sigma-Aldrich (St. Louis, MO). [1-14C]oleic (58.2 mCi/mmol) D-[14C-(U)]glucose (2.9 PerkinElmer NEN® (Boston, Liberase Blendzyme 3 (0.038 WU/ml) Complete Protease Inhibitor Cocktail Roche (Basel, Switzerland). Culture plates flasks Corning (Corning, NY). Glass-bottom six-well MatTek (Ashland, Ninety Scintiplate®, UniFilter® micro plate, Isoplate® scintillation OptiPhase Supermix (Shelton, CT). Ultracentrifugation tubes Beckman Coulter Inc. (#344062; Brea, CA). TLC (Silica gel 60) Merck Millipore (Billerica, Atglistatin Xcess (San Diego, CAY10499 Cayman Chemical (Ann Arbor, MI). TG PAP 150-kit (#61236) BioMerieux (Marcy l'Etoile, France). SYBR Master Mix Kapa Biosystems (Wilmington, Criterion Mini-Protean® TGX™ gels (4–20%), Clarity™ Western ECL Substrate, goat anti-rabbit HRP-conjugated (HRP) secondary antibody #1706515 Bio-Rad (Copenhagen, Pierce™ BCA assay kit (Rockford, IL). Antibodies total thymoma viral proto-oncogene/AKT serine/threonine kinase (Akt) (#9272, rabbit polyclonal antibody), phosphorylated (pSer473, #9271, Slc2a1/Glut1 (#12938, monoclonal Pkm (#3106, Pdha1 (#3205, β-actin (#4970, antibody) Signaling Technology (Beverley, Antibody Pdha1-p300 (#ABS194, Millipore. muscle-associated phosphorylase (Pygm) (#ab81901, Pdk4 (#214938, Abcam (Cambridge, UK). Gapdh (#sc-25778, Santa Cruz Biotechnology (Dallas, TX). #111-035-144 Jackson ImmunoResearch (Suffolk, All chemicals used standard commercial high-purity quality. BAC clone containing genetic locus (AB2.2 ES cells, strain 129S7/SvEvBrd-Hprtb-m2, #bMQ-28H12) modified recombineering generate floxed targeting vector. Because rapidly absence (31Xu Lu Gan Post-translational ubiquitin/proteasome pathway.J. 280: 42841-42847Abstract (208) LoxP sites inserted intron 6 flank exon sequences surfaces. With design, any potential truncated sequence translated genetically expected untarget degraded. details generation vector will published elsewhere (Y.K.L., K.T.D., A.R.K., unpublished observations). Standard homologous recombination 129/SvJEv embryonic stem (ES) target gene. Resulting chimeric bred C57BL/6J (Jackson Laboratory, Bar Harbor, ME) germline transmission. resulting line mixed B6.129/Sv background crossed expressing MMTV-Cre recombinase Laboratory) null (Plin2−/−). generated compliance guidelines given Animal Care Use Committee National Institutes Health under Division Intramural Research, Institute Diabetes Digestive Kidney Diseases, approved animal study protocol K039-LCDB. housed temperature-controlled (22°C) facility strict 12-h light/dark Male backcrossed 10 generations C57BL/6N (Janvier Labs, Le Genest-Saint-Isle, France) chow (#RM3-801190, SDS diets, consisting 12% calories fat, 27% 61% carbohydrate) until 12 weeks age. Mice euthanized cervical dislocation 8:00 10:00 AM, snap-frozen liquid nitrogen −80°C further analysis. use registered Norwegian Research Authority (NARA, protocols FOTS IDs: #6305 #6922) conformed Directive 2010/63/EU European Parliament protection animals scientific purposes. Female siblings (14 weeks) six primary satellite isolation hind leg musulus gastrocnemius musculus soleus. Primary (myoblasts) isolated purified, previously (32Hessvik N.P. Boekschoten M.V. Baltzersen Kersten Andersen Rustan A.C. Thoresen G.H. LXR{beta} dominant LXR subtype lipogenesis cholesterol efflux.Am. 298: E602-E613Crossref (27) Briefly, incubated 2% II 37°C 90 min enzymatically dissociate Satellite liberated digestion 30 min. enrich myoblasts, split population DPBS trypsin EDTA. Further enrichment myoblasts preplating 15 collagen-coated flasks. tends leave behind stick quickly, predominantly fibroblasts. Fibroblasts tend very flat grown collagen, compact smaller diameter. F-10-based medium gives advantage over myotube considered fibroblasts longer visible cultures. Purified proliferated plasticware coated collagen (0.01%). enhance formation, (diluted 1:50 DMEM) seeded differentiate Mouse subconfluence DMEM/Ham's F-10 (1:1, supplemented 20% 5 ng/ml 25 IU penicillin, µg/ml streptomycin, 1.25 μg/ml B 5% CO2. At ∼80% confluence, replaced serum antibiotics induce fusion multinucleated Unless otherwise stated, 4 days experiments. OA FA-free OA:BSA ratio 2.5:1 all experiments (further referred OA). Control OA-stimulated received same concentration BSA. inhibitors (Atglistatin CAY10499) solved DMSO diluted final 0.1% DMSO. inhibitor-stimulated measure response, (100 μM) 24 h. Myotubes glucose-free h presence μM), 15-min incubation nM). wells (six-well plate) pooled RIPA buffer, cocktail, Protein extracts measured kit. sonicated briefly, (33Bindesbøll Berg Fatty acids perilipin5 activating PPARδ.J. 54: 1949-1963Abstract (47) Proteins (15 μg) Laemmli buffer separated 4%–20% gels, blotting nitrocellulose PVDF membranes. Membranes immunoblotted following antibodies: anti-mouse [(31Xu Scholar); #N510; µg/ml], Plin3 [(34Miura J.W. Brzostowski Parisi Schultz C.J. Oliver Functional conservation association Perilipin, ADRP, TIP47 (PAT)-related Drosophila, Dictyostelium.J. 32253-32257Abstract (304) [(33Bindesbøll anti-human transporter 1 (Slc2a1/Glut1, 1:1,000), pyruvate (Pkm, Pygm (1:1,000), dehydrogenase (Pdk4, α (Pdha1, (Pdha1-p300, (detects isoforms Akt1-3, Ser473-phosphorylated GAPDH (Gapdh, 1:500), HRP-labeled antibodies (∼1:10,000). Immunoreactive bands detected visualized using Chemidoc XRS (Bio-Rad), quantified Image Lab software (version 4.0). signal normalize loading. Cells lysed Purification Lysis Solution:PBS (1:1) frozen before isolation. Total RNA preprogrammed "RNA-Cell method." quality quantity determined NanoDrop ND-1000 Spectrophotometer (Thermo Scientific, Waltham, Reverse quantitative real-time PCR (RT-qPCR) performed steps. First, (20 ng/µl) reverse-transcribed single-stranded complementary DNA random hexamers (50°C 60 72°C min). Next, gene-specific regions (70–120 bp) amplified (10 ng) primers nM each) KAPA Fast (10-μl reaction, 95°C min, 40 cycles; s 60°C 20 s) 7900HT instrument (Applied Biosystems, Assay (Tm = designed Primer-BLAST (35Ye Coulouris Zaretskaya I. Cutcutache Rozen Madden T.L. Primer-BLAST: tool design target-specific polymerase chain reaction.BMC Bioinformatics. 134Crossref (3385) primer pairs bind adjacent exons spanned large amplicon sizes ranging 70 110 nt. Primers listed %Table 1. Data analyzed quantification (ΔΔCq method). Resul