Immunofluorescence microscopy of SNAP23 in human skeletal muscle reveals colocalization with plasma membrane, lipid droplets, and mitochondria. Issue 1 (5th January 2016)

• Record Type: Journal Article
• Authors: Strauss, Juliette A.; Shaw, Christopher S.; Bradley, Helen; Wilson, Oliver J.; Dorval, Thierry; Pilling, James; Wagenmakers, Anton J. M.
• Is Part Of: Physiological reports. Volume 4:Issue 1(2016:Jan.)
• Page Start: n/a
• Page End: n/a
• Publication Date: 2016-01-05
• Subjects: Glucose transporter 4 -- intramuscular triglyceride -- lipid droplets -- mitochondria -- synaptosomal‐associated protein 23; Physiology -- Periodicals; 571
• DOI: 10.14814/phy2.12662
• Languages: English
• ISSNs: 2051-817X

Abstract

Abstract: Synaptosomal‐associated protein 23 (SNAP23) is a SNARE protein expressed abundantly in human skeletal muscle. Its established role is to mediate insulin‐stimulated docking and fusion of glucose transporter 4 (GLUT4) with the plasma membrane. Recent in vitro research has proposed that SNAP23 may also play a role in the fusion of growing lipid droplets (LDs) and the channeling of LD‐derived fatty acids (FAs) into neighboring mitochondria for β ‐oxidation. This study investigates the subcellular distribution of SNAP23 in human skeletal muscle using immunofluorescence microscopy to confirm that SNAP23 localization supports the three proposed metabolic roles. Percutaneous biopsies were obtained from the m. vastus lateralis of six lean, healthy males in the rested, overnight fasted state. Cryosections were stained with antibodies targeting SNAP23, the mitochondrial marker cytochrome c oxidase and the plasma membrane marker dystrophin, whereas intramuscular LDs were stained using the neutral lipid dye oil red O. SNAP23 displayed areas of intense punctate staining in the intracellular regions of all muscle fibers and continuous intense staining in peripheral regions of the cell. Quantitation of confocal microscopy images showed colocalization of SNAP23 with the plasma membrane marker dystrophin (Pearson's correlation coefficient r = 0.50 ± 0.01). The intense punctate intracellular staining colocalized primarily with the mitochondrial marker cytochrome C oxidase ( r = 0.50 ± 0.012) and to a lesser extent with LDs ( r = 0.21 ± 0.01) visualized with oil red O. We conclude that the observed subcellular distribution of SNAP23 in human skeletal muscle supports the three aforementioned metabolic roles. Abstract : SNAP23 has an established role in insulin stimulated docking and fusion of GLUT4 with the plasma membrane, while recent in vitro cell culture research suggests that it may also play a role in lipid droplet fusion and channeling of fatty acids released from lipid droplets into mitochondria for beta‐oxidation. This study is the first to investigate the subcellular distribution of SNAP23 in human skeletal muscle in vivo using confocal immunofluorescence microscopy. Colocalisation analysis of muscle cross‐sections labelled with anti‐dystrophin, anti‐COX and oil red O shows that SNAP23 has the highest prevalence at the plasma membrane and mitochondria and a lower prevalence at lipid droplets, giving experimental support for each of the proposed metabolic roles.