206 perilipin proteins are involved in accumulation and metabo- lism of lipids in LDs (Bickel et al. 2009; Brasaemle 2007). Perilipin 5 (PLIN5) expression is highest in tissues with a high fat-oxidative capacity, such as heart, oxidative (type I) skeletal muscles and brown adipose tissue (Yamaguchi et al. 2006; Wolins et al. 2006; Dalen et al. 2007). Recently, we showed that in rat and human skeletal muscle, PLIN5 protein content paralleled the LD content, with most abundant expression in muscle Wbres that have highest fat content (type 1 Wbres in humans, type 2a in rodents) (Minn- aard et al. 2009). PLIN5 has been found covering the lipid droplet and to be present in the cytosol (Wolins et al. 2006; Yamaguchi et al. 2006). PLIN5 overexpression in vitro in OP-9 and COS-7 cells increased cellular triglyceride storage in paral- lel with increases in fatty acid (FA) oxidation and induction of gene expression of mitochondrial enzymes involved in oxidative metabolism (Wolins et al. 2006). These results suggest involvement of PLIN5 in triglyceride storage as well as oxidative degradation of FAs released from the droplet, and suggest involvement of PLIN5 in lipid turn- over. Recently, it has been shown that PLIN5 indeed has the potential to modulate the interaction of the major triacylglycerol lipase ATGL with the LD as well as with its co-factor CGI-58 (Lass et al. 2006) in a well orchestrated manner to modulate LD lipolysis (Granneman et al. 2009, 2011; Wang et al. 2011a). To maintain cellular concentrations of cytotoxic FAs low, eVective shuttling of FAs towards mitochondrial oxi- dation should be facilitated. In oxidative tissues this is in part facilitated by the close vicinity, and sometimes even physical interaction of LD with mitochondria. Functional interactions between LDs and mitochondria have long been reported (Blanchette-Mackie et al. 1995; Cohen et al. 2004; Hoppeler 1999; Hoppeler et al. 1973), and evidence is emerging that mitochondria are not only actively control- ling FA levels in their vicinity by directing FAs towards -oxidation, but the presence of the enzymatic machinery to re-esterify FA in mitochondria indicates that mitochon- dria may also have the option to redirect the fatty acids towards triacylglycerol synthesis in LD. Likewise, isoforms of acyl CoA:diacylglycerol acyltransferase (DGAT) (Stone et al. 2009), glycerol 3-phosphate acyl transferases (GPAT) (Hammond et al. 2002; Lewin et al. 2004) and long-chain acyl-CoA synthetases (Ellis et al. 2010) have been identi- Wed in mitochondria. Orchestrated interaction of processes at the LD and the mitochondria is hence of pivotal impor- tance in tissues like cardiac and skeletal muscle and brown adipose tissue where large Xuctuations in fatty acid oxida- tion are prominent. These tissues also typically express PLIN5 as a LD coat protein. Imaging the LDs and PLIN5 by immunoXuorescence revealed that the majority of PLIN5 is localised to the 123 Histochem Cell Biol (2012) 137:205–216 lipid droplet but also that—mainly in fat oxidative type I muscle Wbres—PLIN5 could be readily detected in a staining pattern similar to recognised mitochondrial proteins (Minnaard et al. 2009). This staining pattern, along with the high expression of PLIN5 in tissues with high FA oxidation capacity (Dalen et al. 2007; Wolins et al. 2006; Yamaguchi et al. 2006) and the observation that overexpressing PLIN5 increased both palmitate oxidation and the expression of genes encoding mitochondrial proteins (Wolins et al. 2006) hints towards involvement of PLIN5 in mitochondrial fat oxidation. Likewise, it has recently been suggested that PLIN5 at the LD surface may recruit LD to the mitochon- dria (Wang and Sztalryd 2011). We here show that PLIN5 is present within the mito- chondria in cardiac and skeletal muscle. PLIN5 overexpres- sion in skeletal muscle indicates that lipid droplets and mitochondria need interaction before PLIN5 possesses its facilitating role to augment mitochondrial fat oxidation. Materials and methods PLIN5 antibody The PLIN5 antibody used in this study is directed against amino acids 451–463 of the C-terminus and was derived from Progen (#GP31; guinea pig polyclonal; Progen, Hei- delberg, Germany). This antibody, as well as other avail- able antibodies against PLIN5, was tested in pilot experiments (Western blots and immunogold labelling for electron microscopy) in a variety of cells and tissues known to vary in endogenous PLIN5 protein expression (HEK and C2C12 cells, white adipose tissue, cardiac muscle, glyco- lytic skeletal muscle (EDL), oxidative skeletal muscle (soleus), liver and testis). Thus, PLIN5 expression was highest in oxidative tissues (heart and oxidative skeletal muscle). The antibody used in this study showed the expected tissue distribution, a double band of the expected size, and a low background and speciWc PLIN5 labelling on the transmission electron microscopy pictures. Negative controls using non-immune serum did not show any bands in Westerns nor did it result in immunogold or immunoXu- orescence staining. Cell culture and treatments HEK293 cells (LGC standards, Teddington, Middlesex, United Kingdom) were maintained in DMEM (GlutaMAX low glucose DMEM, Invitrogen, Breda, The Netherlands) containing antibiotics (50 U/ml penicillin and 50 g/ml streptomycin; Gibco-Invitrogen, Rockville, USA) supple- mented with 10% FCS. Cells were transfected with Plin5 plasmid construct (pcDNA3.1-CMV (2 g per well of a