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Immunofluorescence Microscopy of SNAP23 in Human Skeletal Muscle Reveals Colocalization with Plasma Membrane, Lipid Droplets, and Mitochondria

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Immunofluorescence Microscopy of SNAP23 in Human Skeletal Muscle Reveals Colocalization with Plasma Membrane, Lipid Droplets, and Mitochondria LJMU Research Online Strauss, JA, Shaw, CS, Bradley, H, Wilson, OJ, Dorval, T, Pilling, J and Wagenmakers, AJM Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria. http://researchonline.ljmu.ac.uk/id/eprint/2671/ Article Citation (please note it is advisable to refer to the publisher’s version if you intend to cite from this work) Strauss, JA, Shaw, CS, Bradley, H, Wilson, OJ, Dorval, T, Pilling, J and Wagenmakers, AJM (2016) Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria. Physiological Reports, 4 (1). ISSN 2051-817X LJMU has developed LJMU Research Online for users to access the research output of the University more effectively. Copyright © and Moral Rights for the papers on this site are retained by the individual authors and/or other copyright owners. Users may download and/or print one copy of any article(s) in LJMU Research Online to facilitate their private study or for non-commercial research. You may not engage in further distribution of the material or use it for any profit-making activities or any commercial gain. The version presented here may differ from the published version or from the version of the record. Please see the repository URL above for details on accessing the published version and note that access may require a subscription. For more information please contact [email protected] http://researchonline.ljmu.ac.uk/ http://researchonline.ljmu.ac.uk/ Physiological Reports ISSN 2051-817X ORIGINAL RESEARCH Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria Juliette A. Strauss1, Christopher S. Shaw2, Helen Bradley3, Oliver J. Wilson4, Thierry Dorval5, James Pilling5 & Anton J. M. Wagenmakers1 1 Research Institute for Sport and Exercise Sciences, Liverpool John Moores University, Liverpool, UK 2 Centre for Physical Activity and Nutrition Research, School of Exercise and Nutrition Sciences, Deakin University, Geelong, Victoria, Australia 3 School of Sport, Exercise and Rehabilitation Sciences, University of Birmingham, Edgbaston, UK 4 Institute for Sport, Physical Activity and Leisure, Carnegie Faculty, Leeds Beckett University, Leeds, UK 5 Discovery Sciences, AstraZeneca R&D, Cambridge, UK Keywords Abstract Glucose transporter 4, intramuscular triglyceride, lipid droplets, mitochondria, Synaptosomal-associated protein 23 (SNAP23) is a SNARE protein expressed synaptosomal-associated protein 23. abundantly in human skeletal muscle. Its established role is to mediate insu- lin-stimulated docking and fusion of glucose transporter 4 (GLUT4) with the Correspondence plasma membrane. Recent in vitro research has proposed that SNAP23 may Anton J. M. Wagenmakers, Research Institute also play a role in the fusion of growing lipid droplets (LDs) and the channel- for Sport and Exercise Sciences, Liverpool ing of LD-derived fatty acids (FAs) into neighboring mitochondria for b-oxi- John Moores University, Byrom Street dation. This study investigates the subcellular distribution of SNAP23 in Campus, Liverpool L3 3AF, UK. Tel: +44 (0) 151 9046269 human skeletal muscle using immunofluorescence microscopy to confirm that Fax: +44 (0) 151 9046284 SNAP23 localization supports the three proposed metabolic roles. Percuta- E-mail: [email protected] neous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with Funding Information antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase The authors are grateful for funding of this and the plasma membrane marker dystrophin, whereas intramuscular LDs study in part by Diabetes UK and in part by were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of Astra Zeneca, UK. intense punctate staining in the intracellular regions of all muscle fibers and Received: 7 September 2015; Revised: 27 continuous intense staining in peripheral regions of the cell. Quantitation of November 2015; Accepted: 30 November confocal microscopy images showed colocalization of SNAP23 with the plasma 2015 membrane marker dystrophin (Pearson’s correlation coefficient r = 0.50 Æ 0.01). The intense punctate intracellular staining colocalized primarily with doi: 10.14814/phy2.12662 the mitochondrial marker cytochrome C oxidase (r = 0.50 Æ 0.012) and to a = Æ Physiol Rep, 4 (1), 2016, e12662, lesser extent with LDs (r 0.21 0.01) visualized with oil red O. We con- doi: 10.14814/phy2.12662 clude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles. Introduction paradox that has provoked much research in muscle physiology is that intramuscular triglycerides (IMTG) Defects in insulin-mediated glucose uptake in skeletal accumulate and are positively associated with insulin muscle contribute to whole-body insulin resistance in resistance in obese, sedentary individuals, but are even obesity and precedes the development of type 2 diabetes. higher in endurance-trained athletes who are highly insu- The mechanisms resulting in obesity-induced skeletal lin sensitive. This discrepancy is called the athlete’s para- muscle insulin resistance are only partially understood. A dox (Goodpaster et al. 2001; van Loon 2004). The ª 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of 2016 | Vol. 4 | Iss. 1 | e12662 the American Physiological Society and The Physiological Society. Page 1 This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. Localization of SNAP23 in Human Skeletal Muscle J. A. Strauss et al. current consensus is that in endurance-trained athletes a muscle and impaired glucose tolerance (Sollner 2007; high capacity for fat oxidation and IMTG resynthesis in Bostrom et al. 2010). the period after exercise prevents the accumulation of Transmission electron microscopy and confocal fatty acid (FA) metabolites, such as long-chain acylCoAs, immunofluorescence microscopy have both shown that diacylglycerols, and ceramides, which have been proposed LDs are localized in close proximity to the mitochondrial to induce insulin resistance and reduce skeletal muscle network in skeletal muscle (Hoppeler 1999; Shaw et al. glucose uptake (Ellis et al. 2000; Itani et al. 2002; Kim 2008). Electron microscopy has also shown that a 7-week et al. 2004; Amati et al. 2011; Dube et al. 2011). Synapto- endurance training protocol in previously untrained indi- somal-associated protein 23 (SNAP23) is an understudied viduals (Tarnopolsky et al. 2007) led to an increased spa- protein in skeletal muscle that may link excessive storage tial contact between mitochondria and LDs. This of lipid droplets (LDs) in obese and sedentary individuals increased spatial contact in trained individuals has been to impairments in insulin-mediated glucose uptake via suggested to aid in the efficient oxidation of FAs liberated multiple mechanisms. from lipid droplets upon lipolysis during exercise as it SNAP23 is one of the 36 SNARE (Soluble N Ethyl- reduces the diffusion distance of the released FAs to the maleimide Sensitive Factor Attachment Protein Receptor) site of b-oxidation. In a more recent study in fibroblasts, proteins (Jahn and Scheller 2006). Each of which share a ablation of SNAP23 resulted in a decreased spatial com- characteristic SNARE “motif” in the form of a conserved plex formation between mitochondria and LDs and sequence of 60–70 amino acids. The SNARE motifs medi- decreased mitochondrial b-oxidation (Jagerstrom et al. ate SNARE complex formation, which are important in 2009). This has led to the suggestion that SNAP23 is many intracellular docking and fusion processes. SNAREs implicated in the formation of functional LD-mitochon- are categorized according to the role they play in mem- dria complexes, specifically helping to shuttle LD-derived brane fusion; vesicle-SNAREs (v-SNAREs) are found on FAs into the mitochondria for b-oxidation (Jagerstrom the transport vesicles and target-SNAREs (t-SNAREs) are et al. 2009). found at the plasma membrane. SNAP23 is a t-SNARE Given the proposed roles of SNAP23 in muscle glu- (for a review see Jahn and Scheller 2006), 210 amino cose uptake, LD fusion, and channeling of FA released acids in length, which is expressed in skeletal muscle. In by LD into mitochondria for b-oxidation, this protein its role as a SNARE protein, SNAP23 is able to bind with seems to play an important role in the mechanisms v-SNAREs, VAMP1 and 2 and also syntaxin1, 2, 3, and 4. that lead to impaired glucose uptake, impaired intra- Together these SNARE proteins play key roles in the muscular lipid metabolism, and insulin resistance/type 2 mechanisms that lead to targeted exocytosis. In line with diabetes (Sollner 2007; Bostrom et al. 2010). The pur- its role as a t-SNARE, SNAP23 along with these other pose of this study was to use an extensively validated SNAREs, has been shown to mediate insulin-mediated antibody targeting SNAP23 to describe for the first time GLUT4 docking and fusion with the plasma membrane the fiber-type distribution and subcellular localization of (Foster et al. 1999; Kawanishi et al. 2000). SNAP23 in human skeletal muscle, in particular in the Lipid
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