Eukaryotic cells store neutral lipids in cytoplasmic lipid droplets 1,2 enclosed in a monolayer of phospholipids and associated proteins 3,4 . These dynamic organelles 5 serve as the principal reservoirs for storing cellular energy and for the building blocks for membrane lipids. Excessive lipid accumulation in cells is a central feature of obesity, diabetes and atherosclerosis, yet remarkably little is known about lipid-droplet cell biology. Here we show, by means of a genome-wide RNA interference (RNAi) screen in Drosophila S2 cells that about 1.5% of all genes function in lipiddroplet formation and regulation. The phenotypes of the gene knockdowns sorted into five distinct phenotypic classes. Genes encoding enzymes of phospholipid biosynthesis proved to be determinants of lipid-droplet size and number, suggesting that the phospholipid composition of the monolayer profoundly affects droplet morphology and lipid utilization. A subset of the Arf1-COPI vesicular transport proteins also regulated droplet morphology and lipid utilization, thereby identifying a previously unrecognized function for this machinery. These phenotypes are conserved in mammalian cells, suggesting that insights from these studies are likely to be central to our understanding of human diseases involving excessive lipid storage.We studied lipid-droplet formation in Drosophila Schneider 2 (S2) cells, a proven system for functional genomic studies with efficient gene inactivation by RNAi 6 . We induced lipiddroplet formation by incubation with 1 mM oleate for 24 h. Staining with 4,4-difluoro-1, 3,5,7, showed that droplet size, number and overall volume were increased (Fig. 1a); cellular triacylglycerol content increased sevenfold (Fig. 1b) HHMI Author Manuscript HHMI Author Manuscript HHMI Author Manuscriptcorresponded to lipid droplets with a red fluorescent protein mCherry 7 fused with lipid storage droplet-1 (LSD1), which localizes exclusively to the surface of lipid droplets 3 (not shown).Imaging this process by time-lapse microscopy of BODIPY-labelled cells after oleate addition (Supplementary Movie 1) showed that droplet formation occurred in steps (Fig. 1c). First, increased numbers of small droplets formed in dispersed locations throughout the cell. Next, droplets increased in size and finally aggregated into one or several large clusters, resembling grapes. Electron microscopy confirmed the tight clustering of the droplets, which were often near the nucleus (Supplementary Fig. 3).To unravel the molecular mechanisms governing this progression of changes during lipiddroplet formation, we performed a genome-wide RNAi screen in S2 cells (Fig. 2a). Images were acquired and examined by two independent observers, who scored them for alterations in droplet number, size and dispersion. The same data were analysed computationally (Supplementary Methods). From visual screening, both observers identified 847 candidate genes with altered lipid-droplet morphology. To verify these genes and to minimize the misidentification of genes from of...

The triglycerides in chylomicrons are hydrolyzed by lipoprotein lipase (LpL) along the luminal surface of the capillaries. However, the endothelial cell molecule that facilitates chylomicron processing by LpL has not yet been defined. Here, we show that glycosylphosphatidylinositol-anchored high-density lipoprotein-binding protein 1 (GPIHBP1) plays a critical role in the lipolytic processing of chylomicrons. Gpihbp1-deficient mice exhibit a striking accumulation of chylomicrons in the plasma, even on a low-fat diet, resulting in milky plasma and plasma triglyceride levels as high as 5000 mg/dl. Normally, Gpihbp1 is expressed highly in heart and adipose tissue, the same tissues that express high levels of LpL. In these tissues, GPIHBP1 is located on the luminal face of the capillary endothelium. Expression of GPIHBP1 in cultured cells confers the ability to bind both LpL and chylomicrons. These studies strongly suggest that GPIHBP1 is an important platform for the LpL-mediated processing of chylomicrons in capillaries.

A deficiency in microsomal triglyceride transfer protein (MTP) causes the human lipoprotein deficiency syndrome abetalipoproteinemia. However, the role of MTP in the assembly and secretion of VLDL in the liver is not precisely understood. It is not clear, for instance, whether MTP is required to move the bulk of triglycerides into the lumen of the endoplasmic reticulum (ER) during the assembly of VLDL particles. To define MTP's role in hepatic lipoprotein assembly, we recently knocked out the mouse MTP gene (Mttp). Unfortunately, achieving our objective was thwarted by a lethal embryonic phenotype. In this study, we produced mice harboring a "floxed" Mttp allele and then used Cre-mediated recombination to generate liver-specific Mttp knockout mice. Inactivating the Mttp gene in the liver caused a striking reduction in VLDL triglycerides and large reductions in both VLDL/LDL and HDL cholesterol levels. The Mttp inactivation lowered apo B-100 levels in the plasma by >95% but reduced plasma apo B-48 levels by only ∼20%. Histologic studies in liver-specific knockout mice revealed moderate hepatic steatosis. Ultrastructural studies of wild-type mouse livers revealed numerous VLDL-sized lipid-staining particles within membrane-bound compartments of the secretory pathway (ER and Golgi apparatus) and few cytosolic lipid droplets. In contrast, VLDL-sized lipid-staining particles were not observed in MTPdeficient hepatocytes, either in the ER or in the Golgi apparatus, and there were numerous cytosolic fat droplets. We conclude that MTP is essential for transferring the bulk of triglycerides into the lumen of the ER for VLDL assembly and is required for the secretion of apo B-100 from the liver.J. Clin. Invest. 103:1287-1298(1999 to play an important role in this first "apo B lipidation" step (1,21,25). The existence of apo B in the rough ER of hepatocytes has been documented by immunoelectron microscopy (16). However, the lipidated apo B particles cannot be seen in the rough ER by routine electron microscopy, even when the thin sections are stained for lipids, because those particles are too small (< 150 Å in diameter) and lipid poor to be resolved by this technique. In a second step, the lipidated apo B molecule is thought to acquire the bulk of its core lipids by fusing with a large, VLDL-sized, apo B-free triglyceride particle (a "second-step" particle) (17,23). The existence of the secondstep triglyceride particles within a special compartment of the smooth ER has been supported by 2 different electron microscopic studies (16,23). Biochemical studies of VLDL assembly have also supported the concept that the bulk of neutral lipids are added to apo B in a second step after its translation is complete (1, 24).The role, if any, of MTP in the formation of second-step lipid particles is unclear. In fact, this issue has recently been highlighted as one of the fundamental problems in understanding MTP and lipoprotein assembly (15,26). In recent years, several groups have tried to address this issue by performing metabolic la...

Maintenance of vascular integrity is critical for homeostasis, and temporally and spatially regulated vascular leak is a central feature of inflammation. Sphingosine-1-phosphate (S1P) can regulate endothelial barrier function, but the sources of the S1P that provide this activity in vivo and its importance in modulating different inflammatory responses are unknown. We report here that mutant mice engineered to selectively lack S1P in plasma displayed increased vascular leak and impaired survival after anaphylaxis, administration of platelet-activating factor (PAF) or histamine, and exposure to related inflammatory challenges. Increased leak was associated with increased interendothelial cell gaps in venules and was reversed by transfusion with wild-type erythrocytes (which restored plasma S1P levels) and by acute treatment with an agonist for the S1P receptor 1 (S1pr1). S1pr1 agonist did not protect wild-type mice from PAF-induced leak, consistent with plasma S1P levels being sufficient for S1pr1 activation in wild-type mice. However, an agonist for another endothelial cell G i -coupled receptor, Par2, did protect wild-type mice from PAF-induced vascular leak, and systemic treatment with pertussis toxin prevented rescue by Par2 agonist and sensitized wild-type mice to leak-inducing stimuli in a manner that resembled the loss of plasma S1P. Our results suggest that the blood communicates with blood vessels via plasma S1P to maintain vascular integrity and regulate vascular leak. This pathway prevents lethal responses to leak-inducing mediators in mouse models. IntroductionSphingosine-1-phosphate (S1P), a lipid phosphate produced in the course of sphingosine metabolism in all cell types (1), promotes endothelial cell spreading and barrier function in cell culture (2-5) and in vivo (6, 7). S1P can regulate cell behavior via 5 GPCRs, designated S1P receptor 1 (S1pr1) through S1pr5 (also known as S1P 1 -S1P 5 ) (1,4,8). Models of receptor-dependent roles for S1P in regulating endothelial barrier function have focused on S1P produced by the endothelial cells themselves, casting S1P as a downstream, autocrine/paracrine mediator of the barrier-protective effects of other agents such as activated protein C (9, 10) and angiopoietin (7). However, S1P is present at high concentrations in plasma (11), and the importance of this source of S1P in regulating vascular integrity has not been examined. In addition, GPCR-independent S1P signaling mechanisms and cell-autonomous metabolic effects of disrupting sphingosine conversion to S1P have been reported and may affect vascular integrity (1-5, 7, 12, 13). Central to understanding the physiological roles of S1P in regulating blood vessel function are identification of the sources of S1P that are important for barrier protection in vivo as well as determination of the importance of S1P from blood acting in trans on endothelial cells by receptor-dependent mechanisms ver-

The importance of cholesterol ester synthesis by acyl CoA:cholesterol acyltransferase (ACAT) enzymes in intestinal and hepatic cholesterol metabolism has been unclear. We now demonstrate that ACAT2 is the major ACAT in mouse small intestine and liver, and suggest that ACAT2 deficiency has profound effects on cholesterol metabolism in mice fed a cholesterol-rich diet, including complete resistance to diet-induced hypercholesterolemia and cholesterol gallstone formation. The underlying mechanism involves the lack of cholesterol ester synthesis in the intestine and a resultant reduced capacity to absorb cholesterol. Our results indicate that ACAT2 has an important role in the response to dietary cholesterol, and suggest that ACAT2 inhibition may be a useful strategy for treating hypercholesterolemia or cholesterol gallstones.