Histochem Cell Biol (2012) 137:205–216 Fig. 5 Abundance of mitochon- drial proteins upon PLIN5 over- expression. Western blots were performed for expression of PGC1 (a), VDAC (b) and Wve distinct structural components of the OXPHOS complexes (c) in lysates from whole muscle homogenates (n = 14). Error bars represent SEM, *P < 0.01. The samples were derived from the same experiment and were simultaneously processed in random order A C B 213 as in cardiac muscle. Both subsarcolemmal mitochondria and intramyoWbrillar mitochondria contained PLIN5. Gene electroporation mediated overexpression of PLIN5 doubled PLIN5 protein content and resulted in more numerous and apparently larger LDs in intimate interaction with mito- chondria. Functional assays revealed no direct involvement of PLIN5 in mitochondrial respiration, as respiration in iso- lated mitochondria was not aVected by PLIN5 overexpres- sion. Interestingly though, fatty acid oxidation rates were higher in whole muscle homogenates of PLIN5 over- expressing muscles, suggesting a facilitating role of PLIN5 in augmenting fat oxidation situations when LDs are physi- cally interconnected to the mitochondrial reticulum, possi- bly by shuttling or chaperoning of LD-derived FAs towards mitochondria for oxidation. Recent reports have shown that PLIN5 localised to LDs in tissues rich in mitochondria (Dalen et al. 2007; Wolins et al. 2006; Yamaguchi et al. 2006), that overexpressing PLIN5 in OP-9 and COS-7 cells promoted fat oxidation (Wolins et al. 2006) and that the proteome of LDs con- tained multiple mitochondrial proteins (Brasaemle et al. 2004). In addition, PLIN5 interaction with ATGL on LDs and on undeWned intracellular structures lacking neutral lip- ids (Granneman et al. 2011) has been reported. This has led us to investigate the possibility that PLIN5 also localizes to mitochondria. Thus, we observed that PLIN5 protein content in vastus lateralis muscle of healthy young male subjects correlated positively and tightly with structural components of all Wve complexes of the electron transport chain, but not with mitochondrial proteins without a recognised role in fat oxi- dation. The lack of associations of PLIN5 with VDAC, a mitochondrial protein not involved in (fat) oxidation, nar- rows the interpretation of previous observations of PLIN5 being abundant in mitochondria rich tissues (Dalen et al. 2007; Wolins et al. 2006; Yamaguchi et al. 2006) and may hint towards a more direct involvement of PLIN5 in mito- chondrial fat oxidation. Hence, we studied ADP-driven res- piration rates on palmitoyl-CoA in mitochondria isolated from tibialis anterior muscles of ZDF rats with a range in myocellular fat content. We previously observed that mus- cles of these rats have increased PLIN5 and PLIN2 protein content which tracked with intramyocellular fat content (Minnaard et al. 2009). Fascinatingly, coupled respiration and maximal respiration on palmitoyl-CoA correlated tightly with muscle PLIN5 content but interestingly not with PLIN2 content. Thus, only PLIN5 paralleled changes in mitochondrial fat oxidative capacity. Upon Western blot- ting isolated mitochondria with a speciWc PLIN5 antibody, we were able to unmask PLIN5 as a mitochondrial protein. The mitochondrial isolation protocol used results in a very clean mitochondrial homogenate without contamination of other membrane structures of remnants of LD membranes. This was substantiated by the observation that the mito- chondrial isolation was negative for PLIN2, while showing clear PLIN5 bands in mitochondria isolated from a variety 123