Figure 4. Pqbp-1.1-Venus fusion protein expression in adult nematode. Expression in the pharynx decreases. Intestinal cells keep a high expression level of pqbp-1.1-Venus in adult worms. Arrowheads indicate intestinal cells. Somatic gonads show no expression of pqbp- 1.1-Venus. In the tail region, fluorescence was detected in several neurons (arrows). doi:10.1371/journal.pone.0004104.g004 growth, feeding or lifespan of C. elegans [17]. The Nile Red signals in intestinal cells were clearly lower in homozygous tm3004 than in wild type N2 (Fig. 6A, B) although the number of intestinal cells did not change (Fig. 6A). To confirm these semi-quantitative results regarding lipid contents, we investigated the uptake of exogenous radio-labeled fatty acid (16:0) into various types of lipids (Fig. 7A, B) and the total content of triglyceride (Fig. 7C) in the homozygous tm3004 mutants. The tm3004 mutant shows a decrease in uptake of fatty acid specifically into triglyceride, while the uptake into other phospholipids was not affected. The total content of triglyceride was also decreased (Fig. 7C). These results supported that the T21D12.3 mutation caused reduction of triglyceride, and suggest that the reduction was induced by either impaired synthesis or enhanced degradation of triglyceride. Depletion of PQBP1 paralogue does not lead to reduction of lipid storage In addition to T21D12.3, the genome of C. elegans encodes another paralogue, T07A9.1 (pqbp-1.2), possessing a low homol- ogy to PQBP1 but lacking WWD. Thus, we similarly screened and obtained a mutant of T07A9.1 (tm3045) lacking a part of the gene (Fig. 5B). However, the mutant nematodes did not show reduced signals in Oil Red staining (Fig. 6A), abnormal uptake of radio- labeled fatty acid (16:0) into triglyceride (Fig. 7A, B) or reduction of triglyceride contents (Fig. 7C). Therefore, pqbp-1.1 but not pqbp-1.2 seems to be a proper orthologue of PQBP1 at least from the aspects of molecular structure and lipid metabolism. PQBP1 is involved in lipid metabolism of mammalian white adipocytes We finally tested whether findings in C. elegans were applicable to mammalian adipocytes. Reviewing our previous in situ hybridization data confirmed expression of PQBP1 in fat tissues [14]. Primary white adipocytes prepared from newborn mice were PQBP1 Affects Lipid Storage transfected during growth phase with a plasmid PQBP1-shRNA- ZsGreen, which express both mouse PQBP1-shRNA and ZsGreen. We also used non-silencing shRNA-ZsGreen transfected and non-transfected cells as controls. PQBP1-shRNA actually reduced PQBP1 protein in western blots with anti-PQBP1 antibodies (Fig. 8A). We observed lipid granules were decreased in PQBP1-shRNA expressing fluores- cence-positive cells (arrows) in comparison to fluorescence- negative cells (arrowheads) in the same plate. Both the sizes and Oil Red staining intensities of lipid droplets were significantly reduced (Fig. 8B). Non-silencing shRNA / ZsGreen-expressing cells also did not show any significant change (Fig. 8B). These results collectively support that mammalian PQBP1 also decrease the lipid content. Discussion Nematode PQBP1 homologue is involved in lipid metabolism The method used in this study, which detects a marker fusion protein (PQBP1-Venus) expression under the control of the endogenous enhancer/promoter, definitely shows the endogenous expression pattern of PQBP1 in C. elegans. The expression pattern of nematode PQBP1 was actually unexpected, since we had expected to observe a neuron-dominant expression pattern. However it is not rare that a molecule is differentially used in different organisms even if it is highly conserved beyond species. Moreover, PQBP1 is highly conserved in Arabidopsis although the plant does not have the brain. Our results clearly showed that nematode pqbp-1.1 is expressed in cells for lipid-storage (Fig. 1–4) and that pqbp-1.1 depletion links to abnormal lipid metabolism in nematode (Fig. 5, 6). Mammals possess fat cells to store lipid in the body, while nematode keep lipid in intestinal cells. However, although the cell types for lipid storage are different, compositions of lipid (such as saturated or unsaturated fatty acid, triglyceride) and metabolic pathways are fundamentally conserved beyond species. In addition, the knock down experiment with shRNA showed reduction of lipid storage in mammalian white adipocyte (Fig. 7). Therefore, the mutant nematodes generated in this study could be a hypothetical model for the lean body symptom of human patients linked to PQBP1 mutations. This model will be available for further analysis of the detailed molecular mechanisms underlying the lean body, and such studies might develop PQBP1 to a novel target molecule in human lipid metabolism. Lipid metabolism in PQBP1 and polyQ pathologies PQBP1 is known to interact with mutant Htt [18]. Meanwhile, mutant huntingtin (Htt), the causative gene product of Hunting- ton’s disease (HD), is known to downregulate peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC- 1a) [19–21], a co-activator of peroxisome proliferator-activated receptor (PPAR-c) that is essential for transcription of a number of genes involved in glucose metabolism and b-oxidation of fatty acid [for review see 22]. Interestingly, in addition to binding to the promoter, PGC-1a participates in RNA elongation and splicing through binding to factors anchored onto C-terminal domain of RNA polymerase II [22]. The binding property of PGC-1a to RNA polymerase II and splicing proteins is homologous to that of PQBP1 [2,4], suggesting a possible relationship between PQBP1 and PGC-1a. Therefore, it might be worth analyzing whether PQBP1 is involved in the same transcription protein complex with PGC-1a and how PQBP1 affects lipid metabolism mediated by PGC-1a in normal physiology and in HD pathology. PLoS ONE | www.plosone.org 4 January 2009 | Volume 4 | Issue 1 | e4104