212 Histochem Cell Biol (2012) 137:205–216 Fig. 3 PLIN5 overexpression results in large LDs. Left hand panels a and c show empty vector control data, right hand panels b and d show PLIN5 overexpression data. Note the apparent larger LD size upon PLIN5 overexpression. Bar represents 10 m in the upper panel and 2 m in the lower panels not reveal signiWcant diVerences in respiration, neither on pyruvate (empty vector vs. PLIN5 15.98 § 1.17 vs. 15.48 § 1.18 state 3/state 2, P = 0.83; 22.53 § 0.88 vs. 22.29 § 1.44 state U/state 2, P = 0.68), nor on palmitoyl- CoA (empty vector vs. PLIN5 6.76 § 0.38 vs. 7.36 § 0.30 state 3/state 2, P = 0.23; 8.56 § 0.63 vs. 9.14 § 0.49 state U/state 2, P = 0.37) (Fig. 6a, b). Thus, in a system in which PLIN5-coated LDs are dissociated from the mitochondria, PLIN5 overexpression does not augment mitochondrial (fat) oxidative capacity. PLIN5 overexpression improved 14C-palmitate oxidation Given the intimate association of LDs with mitochondria we performed complementary measurements of fat oxida- tion using 14C-palmitate in whole muscle homogenates when LDs and mitochondria were not dissociated. Total 14C-palmitate oxidation was not changed (empty vector 0.71 § 0.06 nmol/2 h/mg protein, PLIN5 0.71 § 0.03 nmol/2 h/mg protein, P = 0.99) (Fig. 6c). However, oxida- tion of 14C-palmitate to 14CO2 increased by 44.8% (empty vector 0.05 § 0.008, PLIN5 0.07 § 0.012 14CO2/total 14C-palmitate oxidation, P = 0.05) (Fig. 6d). The respira- tion data in isolated mitochondria and in muscle homoge- nates jointly indicate that overexpression of PLIN5 does not augment mitochondrial fat oxidative capacity per se but rather creates a situation in which shuttling of FAs towards 123 Fig. 4 PLIN5 labelling in mitochondria and on the lipid droplets. Immunogold staining of PLIN5 in tibialis anterior muscle of empty vec- tor (control) (a) or PLIN5-electroporated muscle (b). PLIN5 overexpression b, c and d resulted in larger lipid droplets and intimate interaction of lipid droplets with mitochondria at PLIN5 positive sites (see arrowheads in c and d). Bars represent 1 m. e Intramitochondrial PLIN5 labelling. Bar represents 0.5 m mitochondria becomes more eYcient, thereby protecting mitochondria against the deleterious eVects of lipid overXow. Discussion PLIN5 was recently identiWed as a member of the perilipin family of lipid droplet coating proteins and was considered to be an exchangeable LD coat protein; unlike perilipins 1 and 2, PLIN5 is not exclusively found in the LD coat but also cytosolically (Dalen et al. 2007; Wolins et al. 2006; Yamaguchi et al. 2006). Using a variety of validated tech- niques, however, we here show that PLIN5 in muscle is found at the LDs as well as in mitochondria, this in contrast to PLIN2, the other major LD coat protein found in muscle. PLIN5 positive mitochondria were detected in oxidative (soleus), glycolytic (EDL), and mixed (TA) muscles as well