Fsp27, a member of the Cide family proteins, was shown to localize to lipid droplet and promote lipid storage in adipocytes. We aimed to understand the biological role of Fsp27 in regulating adipose tissue differentiation, insulin sensitivity and energy balance. Fsp27 −/− mice and Fsp27/lep double deficient mice were generated and we examined the adiposity, whole body metabolism, BAT and WAT morphology, insulin sensitivity, mitochondrial activity, and gene expression changes in these mouse strains. Furthermore, we isolated mouse embryonic fibroblasts (MEFs) from wildtype and Fsp27 −/− mice, followed by their differentiation into adipocytes in vitro. We found that Fsp27 is expressed in both brown adipose tissue (BAT) and white adipose tissue (WAT) and its levels were significantly elevated in the WAT and liver of leptin-deficient ob/ob mice. Fsp27 −/− mice had increased energy expenditure, lower levels of plasma triglycerides and free fatty acids. Furthermore, Fsp27 −/− and Fsp27/lep double-deficient mice are resistant to diet-induced obesity and display increased insulin sensitivity. Moreover, white adipocytes in Fsp27 −/− mice have reduced triglycerides accumulation and smaller lipid droplets, while levels of mitochondrial proteins, mitochondrial size and activity are dramatically increased. We further demonstrated that BAT-specific genes and key metabolic controlling factors such as FoxC2, PPAR and PGC1α were all markedly upregulated. In contrast, factors inhibiting BAT differentiation such as Rb, p107 and RIP140 were down-regulated in the WAT of Fsp27 −/− mice. Remarkably, Fsp27 −/− MEFs differentiated in vitro show many brown adipocyte characteristics in the presence of the thyroid hormone triiodothyronine (T3). Our data thus suggest that Fsp27 acts as a novel regulator in vivo to control WAT identity, mitochondrial activity and insulin sensitivity.

Unesterified sterol modulates the function of eukaryotic membranes. In human cells, sterol is esterified to a storage form by acyl-coenzyme A (CoA): cholesterol acyl transferase (ACAT). Here, two genes are identified, ARE1 and ARE2, that encode ACAT-related enzymes in yeast. The yeast enzymes are 49 percent identical to each other and exhibit 23 percent identity to human ACAT. Deletion of ARE2 reduced sterol ester levels to approximately 25 percent of normal levels, whereas disruption of ARE1 did not affect sterol ester biosynthesis. Deletion of both genes resulted in a viable cell with undetectable esterified sterol. Measurements of [14C]acetate incorporation into saponified lipids indicated down-regulation of sterol biosynthesis in the are1 are2 mutant cells. With the use of a consensus sequence to the yeast and human genes, an additional number of the ACAT gene family was identified in humans.

Mature white adipocytes contain a characteristic unilocular lipid droplet. However, the molecular mechanisms underlying unilocular lipid droplet formation are poorly understood. We previously showed that Fsp27, an adipocyte-specific lipid droplet-associated protein, promotes lipid droplet growth by initiating lipid exchange and transfer. Here, we identify Perilipin1 (Plin1), another adipocyte-specific lipid droplet-associated protein, as an Fsp27 activator. Plin1 interacts with the CIDE-N domain of Fsp27 and markedly increases Fsp27-mediated lipid exchange, lipid transfer and lipid droplet growth. Functional cooperation between Plin1 and Fsp27 is required for efficient lipid droplet growth in adipocytes, as depletion of either protein impairs lipid droplet growth. The CIDE-N domain of Fsp27 forms homodimers and disruption of CIDE-N homodimerization abolishes Fsp27-mediated lipid exchange and transfer. Interestingly, Plin1 can restore the activity of CIDE-N homodimerization-defective mutants of Fsp27. We thus uncover a novel mechanism underlying lipid droplet growth and unilocular lipid droplet formation that involves the cooperative action of Fsp27 and Plin1 in adipocytes.

Lipid droplets (LDs) are intracellular organelles with neutral lipid cores surrounded by a phospholipid monolayer and coated with various proteins ( 1-3 ). LDs have been found in almost all eukaryotic organisms from yeast to mammals ( 4 ). They interact with other cellular organelles ( 5-8 ), and their dynamics is closely related to the progression of metabolic diseases, such as obesity, fatty liver, type 2 diabetes mellitus, and atherosclerosis ( 9 ). Recent studies have also shown that LDs are involved in the reproduction of infectious hepatitis C virus particles ( 10 ) and in protecting cells from damage ( 11 ). The identifi cation of perilipin, ADRP, and Tip47 (PAT) family proteins has provided useful marker proteins to facilitate the purifi cation of LDs. Recent proteomic studies suggesting that LDs are not simply inert cellular inclusions for the storage of neutral lipids, but rather functional cellular organelles, has established a new era in LD research ( 3,(12)(13)(14)(15)(16)(17)(18).Although LDs are highly dynamic organelles involved in many cellular activities, especially lipid metabolism, the molecular mechanisms that govern LD formation remain largely unknown. The current model of LD biogenesis speculates that LDs are derived from the endoplasmic reticulum (ER) by a process that begins with the accumulation of neutral lipids between the leafl ets of phospholipid bilayers ( 3,19 ). Many studies have attempted to unravel how LDs form and grow, but this hypothesis still lacks direct evidence and the molecular mechanism Abstract Storage of cellular triacylglycerols (TAGs) in lipid droplets (LDs) has been linked to the progression of many metabolic diseases in humans, and to the development of biofuels from plants and microorganisms. However, the biogenesis and dynamics of LDs are poorly understood. Compared with other organisms, bacteria seem to be a better model system for studying LD biology, because they are relatively simple and are highly effi cient in converting biomass to TAG. We obtained highly purifi ed LDs from Rhodococcus sp. RHA1, a bacterium that can produce TAG from many carbon sources, and then comprehensively characterized the LD proteome. Of the 228 LD-associated proteins identifi ed, two major proteins, ro02104 and PspA, constituted about 15% of the total LD protein. (Grant 2009CB919000, Grant 2010CB833703; Grant 2011CBA00900), and the National Natural Science Foundation of China (Grant 30871229, Grant 30971431, and Grant 31000365 Abbreviations: ER, endoplasmic reticulum; LD, lipid droplet; MLDS, microorganism lipid droplet small; MSM, mineral salt medium; NB, nutrient broth; PAT, perilipin, ADRP, and Tip47; PspA, phage shock protein A; TAG, triacylglycerol; TEM, transmission electron microscopy.

The oxysterol-binding protein (OSBP)-related proteins (ORPs) are conserved from yeast to humans, and implicated in the regulation of lipid homeostasis and in signaling pathways. Saccharomyces cerevisiae has seven ORPs (Osh1-Osh7) that share one unknown essential function. Here, we report the 1.5-2.3 Å structures of the PH domain and ORD (OSBP-related domain) of yeast Osh3 in apo-form or in complex with phosphatidylinositol 4-phosphate (PI[4]P). Osh3 recognizes PI(4)P by the highly conserved residues in the tunnel of ORD whereas it lacks sterol binding due to the narrow hydrophobic tunnel. Yeast complementation tests suggest that PI(4)P binding to PH and ORD is essential for function. This study suggests that the unifying feature in all ORP homologs is the binding of PI(4)P to ORD and sterol binding is additional to certain homologs. Structural modeling of full-length Osh3 is consistent with the concept that Osh3 is a lipid transfer protein or regulator in membrane contact sites.