Histochem Cell Biol (2012) 137:205–216 acid derivatives—for mitochondrial oxidation. Measure- ments of respiration in isolated mitochondria did not reveal any diVerences between mitochondria derived from the sham- and the PLIN5-electroporated muscle, which does not support a direct role for PLIN5 in mitochondrial respi- ration. Interestingly though, when examining 14C-palmitate oxidation in muscle homogenates—when mitochondria are not examined in isolation but in context of their native sub- cellular milieu—we observed increased complete oxidation of 14C-palmitate to CO2. This occurred in the absence of an increase in accumulation of acid soluble metabolites, indi- cating that the supply of fatty acids and/or the rate of -oxi- dation were better matched with the capacity for downstream FA oxidative metabolism. It should be noted that, albeit unlikely, we could not exclude the possibility that, during the isolation of mitochondria, the stronger physical interaction between mitochondria and lipid drop- lets upon PLIN5 overexpression resulted in a loss of mito- chondria intimately connected to the large lipid droplets. This could explain the discrepancy in the results of the experiments in muscle homogenates and isolated mitochon- dria. Nevertheless, the increase 14C-palmitate to CO2 points towards a beneWcial eVect of PLIN5 overexpression on mitochondrial fatty acid oxidation. Thus, given the dual localization of PLIN5 to the LD and to mitochondria, it is tempting to speculate that next to a role for PLIN5 in releasing fatty acids from the LD by co-regulat- ing lipolysis, PLIN5 is also involved in shuttling/chaperoning/ binding fatty acids or fatty acid derivatives from LD origin to mitochondria for sake of oxidation. While this suggestion clearly needs further experimental substantiation, it is well in line with previous observations by others that overexpressing PLIN5 augmented fat oxidation (Wolins et al. 2006). In the process of Wnalising this manuscript, Wang et al. (Wang et al. 2011b) showed that PLIN5 is an important mediator of the release of fatty acids for mitochondrial -oxidation in liver cells. Upon PKA stimulation, PLIN5 increases LD hydrolysis thus making fatty acids available for downstream mitochon- drial metabolism (Wang et al. 2011b). Whether the same holds true for muscle deserves further investigation. Although our data clearly support mitochondrial localiza- tion of PLIN5 it remains to be solved how PLIN5 gets into mitochondria. We were unable to Wnd evidence for an N-ter- minal mitochondrial targeting pre-sequence following tar- geted database searches using PSORT (Horton et al. 2007) and SignalP (Dyrløv Bendtsen et al. 2004). Although such a pre-sequence is common for many mitochondrial proteins (van der Laan et al. 2010), it should be noted that a variety of other mitochondrial proteins do not contain such a pre- sequence either (Dukanovic and Rapaport 2011). Rather, the mitochondrial localization of these proteins is determined by structural characteristics of the protein. It has been postulated that protein localization to LDs is determined by basic and 215 hydrophobic domains, with remarkable similarities to the sig- nals that target proteins to mitochondria (Ingelmo-Torres et al. 2009). Interestingly, we did Wnd structural homology of PLIN5 with the alpha and beta chains of the mitochondrial protein ATP synthase (PropSearch) (Hobohm and Sander 1995), providing additional support for structural characteris- tics in the PLIN5 sequence that could determine mitochon- drial localization. Proteome analysis of the LD coat interestingly revealed that the mitochondrial protein ATP synthase was present in LD fractions of lipolytically stimu- lated adipocytes (Brasaemle et al. 2004). Thus, PLIN5 would not be the Wrst protein localised to mitochondria and lipid droplets while lacking a mitochondrial targeting pre- sequence. Moreover, recent work revealed that the last 20 amino acids of the C-terminal end of the protein were identi- Wed to be essential for recruitment of mitochondria to the lipid droplet surface (Wang et al. 2011b). In conclusion, we provide evidence that PLIN5 is abun- dantly expressed in subsarcolemmal and intermyoWbrillar mitochondria of glycolytic, oxidative and cardiac muscle as well as on the interface of mitochondria and lipid droplets. PLIN5 overexpression augmented fat oxidation in muscle homogenates containing both LDs and mitochondria but not in isolated mitochondria. These Wndings unmask PLIN5 as a protein localised to LD and mitochondria with a puta- tive role in channelling LD derived fatty acids towards mitochondrial oxidation. Interaction of PLIN5 coated LD with mitochondria appears more frequent and more inti- mate upon skeletal muscle PLIN5 overexpression. Acknowledgments Research presented in this article was Wnancially supported by NUTRIM and the graduate school VLAG. A VICI (grant 918.96.618) and a VIDI (grant 917.66.359) for innovative research from the Netherlands Organization for ScientiWc Research (NWO) supports the work of P. Schrauwen and M. Hesselink, respectively. P. Bickel’s work was supported by NIH grant DK068046. Open Access This article is distributed under the terms of the Crea- tive Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited. 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