Table 2 Correlation of PLIN5 protein expression with respiration of mitochondria isolated from skeletal muscle of fatty ZDF rats PLIN5 Pearson correlation P n PLIN2 Pearson correlation P n Palmitoyl-CoA State 3 0.713* <0.001 23 0.343 0.1 24 VDAC OXPHOS I OXPHOS II OXPHOS III OXPHOS IV OXPHOS V Pearson correlation P ¡0.148 0.664 0.721* 0.012 0.455 0.159 0.810* 0.003 0.704* 0.016 0.606* 0.048 Histochem Cell Biol (2012) 137:205–216 Table 1 Correlation of PLIN5 protein expression with OXPHOS complexes in human vastus lateralis muscle and processed for standard SDS-PAGE and Western blot- ting. Protein concentration was determined and equal amounts of protein were loaded per lane. The nitrocellulose membranes were incubated with antibodies against PLIN5 (GP31; Progen Biotechnik, Heidelberg, Germany), PLIN2 (GP40; Progen Biotechnik, Heidelberg, Germany), OXPHOS (MS601; MitoSciences, Eugene, OR, USA), PGC1 (516,557; Calbiochem; VWR International BV, Amsterdam, The Netherlands), VDAC (SC-8,828; Santa Cruz Biotechnology, Tebu-bio, Heerhugowaard, The Neth- erlands), and SR-actin (A-2,172; Sigma, St. Louis, USA). Blots incubated with PLIN5, PLIN2, OXPHOS, PGC1 , VDAC and sr-actin were probed with appropriate IRDye800-conjugated or IRDye700-conjugated secondary antibodies (Rockland, Tebu-bio, Heerhugowaard, The Netherlands, and LICOR Biosciences, Westburg, Leusden, The Netherlands), and bands at a molecular weight corre- sponding to the control samples were quantiWed using the Odyssey infrared imaging system (LICOR Biosciences, Westburg, Leusden, The Netherlands). Statistical analysis Results are presented as mean § SE. DiVerences between groups were evaluated with paired t tests. Outcomes were considered statistically signiWcant when P < 0.05 (two- tailed). Pearson’s correlation coeYcients were used to describe the linear association between variables. All analy- ses were performed using the Statistical Package for the Social Sciences (SPSS 16.0, Nieuwegein, The Netherlands) software for Mac 16.0. Results Skeletal muscle PLIN5 protein content correlates with oxidative capacity In order to explore the role of PLIN5 in skeletal muscle, we measured PLIN5 protein content in muscle biopsies of vas- tus lateralis muscle of young healthy male subjects. PLIN5 protein content correlated strongly with the protein content of structural components of the individual complexes of the electron transport chain; OXPHOS complexes I (r = 0.721, P = 0.012), III (r = 0.810, P = 0.003), IV (r = 0.704, P = 0.016) and V (r = 0.606, P = 0.048) but not with protein 209 State U 0.663* 0.001 23 0.182 0.396 24 content of VDAC, an outer mitochondrial membrane pro- tein without a recognised role in (fat) oxidation (r = 0.148, P = 0.664) (n = 11) (Table 1). These observations indi- rectly suggest a role of PLIN5 in mitochondrial fat oxida- tive metabolism. This led us to investigate the relationship between PLIN5 protein content in whole muscle homoge- nates with respiration rates of isolated muscle mitochondria on a fat-derived substrate (palmitoyl-CoA). Thus, we ana- lysed correlations of skeletal muscle PLIN5 protein expres- sion with mitochondrial function in a model of progressive myocellular fat deposition, the maturing Zucker diabetic fatty (ZDF) rats (fa/fa). In this model, PLIN5 protein content tracks with intramyocellular lipid content (Minnaard et al. 2009). We examined the correlation between PLIN5 content in whole muscle homogenates and intrinsic mitochondrial func- tion in mitochondria isolated from these muscles by classical mitochondrial respirometry using palmitoyl-CoA as a FA- derived substrate. In these rats, PLIN5 protein content, but importantly not PLIN2 protein content, positively correlated with ADP-driven state 3 respiration (r = 0.713, P < 0.001) as well as respiration under maximal FCCP-mediated uncoupled conditions (state U, r = 0.663, P = 0.001) (Table 2). These observations may indicate that in muscles in which PLIN5 protein content scales with LD content, PLIN5 is involved in facilitating increased mitochondrial fat oxidation. Perilipin 5 localizes to lipid droplets as well as to mito- chondria in cardiac and skeletal muscle. The strong correla- tion of PLIN5 protein content with OXPHOS complexes and mitochondrial respiration on a FA-derived substrate led us to examine whether PLIN5, next to its recognised locali- sation to LD, is also present in mitochondria. Thus, we 123