214 Histochem Cell Biol (2012) 137:205–216 Fig. 6 EVects of PLIN5 overexpression on mitochondrial respiration and FA oxidation. a and b State 3 (coupled) and state U (uncoupled) respiration on palmitoyl-CoA (a) or pyruvate (b), values are standard- ised to state 2 respiration (background respiration in the absence of ADP) (n = 9). c Total 14C-palmitate oxidation in muscle homogenates (CO2 plus ASMs). d The ratio of CO2 production (complete oxidation) to total FA oxidation (CO2 plus acid soluble metabolites) (n = 7). Error bars represent SEM, P values were derived from paired t tests of skeletal and cardiac muscles. To further substantiate these Wndings we performed double immunoXuorescence microscopy with PLIN5 along with an antibody against structural components of the electron transport chain in rat tibialis anterior muscles. Confocal analysis of tissue sec- tions indeed localised PLIN5 to the LD, but also to the mitochondria. For a more detailed conWrmation of PLIN5 being expressed in mitochondria we applied post-embed- ding immunogold electron microscopy, the gold standard for high-resolution subcellular localisation studies of pro- teins (Zuber et al. 2005). Thus, we identiWed PLIN5 to the LD membrane as well as in mitochondria in sections from rat glycolytic (EDL), oxidative (soleus) and cardiac muscle. PLIN5 was consistently present in subsarcolemmal as well as in intermyoWbrillar mitochondria of all muscle types examined. Again, no such observations were made for PLIN2, the other major LD coat protein in muscle. Except for LDs, other organelles and myoWbrillar structures were almost devoid of any PLIN5 signal. Having identiWed PLIN5 in mitochondria we set out to investigate the eVect of PLIN5 overexpression on PLIN5 subcellular distribution and myocellular ultrastructure. We deliberately choose unilateral gene electroporation as the in 123 vivo approach to overexpress PLIN5 in the tibialis anterior muscle of a rat. Thus, we were able to avoid lifelong adap- tive responses while creating the ability to use the contralat- eral leg as an internal control. This resulted in a two-fold overexpression of PLIN5. ImmunoXuorescence microscopy and immunogold electron microscopy in tibialis anterior samples of the control and the overexpressing muscle con- Wrmed speciWc localisation of PLIN5 to mitochondria and lipid droplets. No apparent changes in the subcellular distri- bution of PLIN5 were observed, i.e., subsarcolemmal and intermyoWbrillar mitochondria pools both remained PLIN5 positive and qualitative examination of electron microscopi- cal images did not indicate selective upregulation of PLIN5 at the LD site or at mitochondria. Using a variety of mito- chondrial proteins we did not obverse indications of increased mitochondrial density upon PLIN5 overexpres- sion during the 8 day time span of PLIN5 overexpression. Moreover, PLIN5 overexpression augmented intramyo- cellular lipid storage and LD size, possibly reXecting the involvement of PLIN5 in ATGL-mediated lipolysis (Granneman et al. 2011; Wang et al. 2011a). The localiza- tion of PLIN5 on the LDs and in mitochondria suggests a role for PLIN5 in directing or binding fatty acids—or fatty