Lipid Droplet Protein Research Articles

Role of adipose differentiation-related protein in lung surfactant production: a reassessment

Based on data developed with the use of isolated lipid droplets from neonatal rat lung lipofibroblasts, we speculated previously that the droplet coat protein, adipose differentiation-related protein (ADFP), mediated the transfer of lipids into type 2 lung epithelial cells for the production of surfactant phospholipids. The present studies were designed to test the role of ADFP in this transfer with the use of ADFP-coated lipid droplets from CHO fibroblast cells and a cultured human lung epithelial cell line. We found no role for ADFP in the lipid transfer and conclude that a lipase associated with the lipid droplets hydrolyzes their core triacylglycerols, releasing fatty acids that are taken up by the epithelial cells.

A proposed model of fat packaging by exchangeable lipid droplet proteins

Humans have evolved mechanisms of efficient fat storage to survive famine, but these mechanisms contribute to obesity in our current environment of plentiful food and reduced activity. Little is known about how animals package fat within cells. Five related structural proteins serve roles in packaging fat into lipid droplets. The proteins TIP47, S3–12, and OXPAT/MLDP/PAT-1 move from the cytosol to coat nascent lipid droplets during rapid fat storage. In contrast, perilipin and adipophilin constitutively associate with lipid droplets and play roles in sustained fat storage and regulation of lipolysis. Different tissues express different complements of these lipid droplet proteins. Thus, the tissue-specific complement of these proteins determines how tissues manage lipid stores.

Identification and Characterization of Associated with Lipid Droplet Protein 1: A Novel Membrane‐Associated Protein That Resides on Hepatic Lipid Droplets

Alcoholic and nonalcoholic liver steatosis and steatohepatitis are characterized by the massive accumulation of lipid droplets (LDs) in the cytosol of hepatocytes. Although LDs are ubiquitous and dynamic organelles found in the cells of a wide range of organisms, little is known about the mechanisms and sites of LD biogenesis. To examine the participation of these organelles in the pathophysiological disorders of steatotic livers, we used a combination of mass spectrometry (matrix-assisted laser desorption ionization-time of flight and LC-MS electrospray) and Western blot analysis to study the composition of LDs purified from rat liver after a partial hepatectomy. Fifty proteins were identified. Adipose differentiation-related protein was the most abundant, but other proteins such as calreticulin, TIP47, Sar1, Rab GTPases, Rho and actin were also found. In addition, we identified protein associated with lipid droplets I ALDI (tentatively named Associated with LD protein 1), a novel protein widely expressed in liver and kidney corresponding to the product of 0610006F02Rik (GI:27229118). Our results show that, upon lipid loading of the cells, ALDI translocates from the endoplasmic reticulum into nascent LDs and indicate that ALDI may be targeted to the initial lipid deposits that eventually form these droplets. Moreover, we used ALDI expression studies to view other processes related to these droplets, such as LD biogenesis, and to analyze LD dynamics. In conclusion, here we report the composition of hepatic LDs and describe a novel bona fide LD-associated protein that may provide new insights into the mechanisms and sites of LD biogenesis.

Structural Determinants That Target the Hepatitis C Virus Core Protein to Lipid Droplets

Hepatitis C virus core protein is targeted to lipid droplets, which serve as intracellular storage organelles, by its C-terminal domain, termed D2. From circular dichroism and nuclear magnetic resonance analyses, we demonstrate that the major structural elements within D2 consist of two amphipathic alpha-helices (Helix I and Helix II) separated by a hydrophobic loop. Both helices require a hydrophobic environment for folding, indicating that lipid interactions contribute to their structural integrity. Mutational studies revealed that a combination of Helix I, the hydrophobic loop, and Helix II is essential for efficient lipid droplet association and pointed to an in-plane membrane interaction of the two helices at the phospholipid layer interface. Aside from lipid droplet association, membrane interaction of D2 is necessary for folding and stability of core following maturation at the endoplasmic reticulum membrane by signal peptide peptidase. These studies identify critical determinants within a targeting domain that enable trafficking and attachment of a viral protein to lipid droplets. They also serve as a unique model for elucidating the specificity of protein-lipid interactions between two membrane-bound organelles.

Recruitment of TIP47 to lipid droplets is controlled by the putative hydrophobic cleft

Adipose differentiation-related protein (ADRP) and TIP47 show sequence similarity, particularly in their N-terminal PAT-1 domain. Under standard culture conditions, ADRP existed in most lipid droplets (LDs), whereas TIP47 was observed only in some LDs and recruited to LDs on treatment with fatty acids. By analyzing deletion mutants, we found that the C-terminal half of TIP47, or more specifically the putative hydrophobic cleft [S.J. Hickenbottom, A.R. Kimmel, C. Londos, J.H. Hurley, Structure of a lipid droplet protein; the PAT family member TIP47, Structure (Camb) 12 (2004) 1199–1207.], was involved in LD targeting and responsiveness to fatty acids. The result contrasted with that observed for ADRP and implied a distinct LD-targeting mechanism for TIP47. Consistent with this, overexpression of Rab18 decreased ADRP, but not TIP47, from LDs, and TIP47 did not displace pre-existing ADRP from LDs. But ADRP may be a factor to control the TIP47 behavior, because TIP47 in LDs increased upon down-regulation of ADRP. The results suggested that the putative hydrophobic cleft is critical for the unique characteristics of TIP47.

Characterization of the Drosophila Lipid Droplet Subproteome

Lipid storage droplets are universal organelles essential for the cellular and organismal lipometabolism including energy homeostasis. Despite their apparently simple design they are proposed to participate in a growing number of cellular processes, raising the question to what extent the functional multifariousness is reflected by a complex organellar proteome composition. Here we present 248 proteins identified in a subproteome analysis using lipid storage droplets of Drosophila melanogaster fat body tissue. In addition to previously known lipid droplet-associated PAT (Perilipin, ADRP, and TIP47) domain proteins and homologues of several mammalian lipid droplet proteins, this study identified a number of proteins of diverse biological function, including intracellular trafficking supportive of the dynamic and multifaceted character of these organelles. We performed intracellular localization studies on selected newly identified subproteome members both in tissue culture cells and in fat body cells directly. The results suggest that the lipid droplets of fat body cells are of combinatorial protein composition. We propose that subsets of lipid droplets within single cells are characterized by a protein "zip code," which reflects functional differences or specific metabolic states.

Proteomic Profiling of Lipid Droplet Proteins in Hepatoma Cell Lines Expressing Hepatitis C Virus Core Protein

Hepatitis C virus (HCV) core protein has been suggested to play crucial roles in the pathogeneses of liver steatosis and hepatocellular carcinomas due to HCV infection. Intracellular HCV core protein is localized mainly in lipid droplets, in which the core protein should exert its significant biological/pathological functions. In this study, we performed comparative proteomic analysis of lipid droplet proteins in core-expressing and non-expressing hepatoma cell lines. We identified 38 proteins in the lipid droplet fraction of core-expressing (Hep39) cells and 30 proteins in that of non-expressing (Hepswx) cells by 1-D-SDS-PAGE/MALDI-TOF mass spectrometry (MS) or direct nanoflow liquid chromatography-MS/MS. Interestingly, the lipid droplet fraction of Hep39 cells had an apparently lower content of adipose differentiation-related protein and a much higher content of TIP47 than that of Hepswx cells, suggesting the participation of the core protein in lipid droplet biogenesis in HCV-infected cells. Another distinct feature is that proteins involved in RNA metabolism, particularly DEAD box protein 1 and DEAD box protein 3, were detected in the lipid droplet fraction of Hep39 cells. These results suggest that lipid droplets containing HCV core protein may participate in the RNA metabolism of the host and/or HCV, affecting the pathopoiesis and/or virus replication/production in HCV-infected cells.

MLDP, a Novel PAT Family Protein Localized to Lipid Droplets and Enriched in the Heart, Is Regulated by Peroxisome Proliferator-activated Receptor α

Cytosolic lipid droplets (LDs) are multifunctional organelles that exist in all types of eukaryotic cells and control lipid homeostasis. In mammalian cells LDs contain a class of proteins in their surface layers that share a homologous sequence called the PAT domain, including perilipin, adipose differentiation-related protein (ADRP), a tail-interacting protein of 47 kDa (TIP47), and S3-12, which are distributed tissue- or cell type-selectively. Expression in some cases is regulated by peroxisome proliferator-activated receptors (PPARs). In this study we identified a new PAT family member named MLDP (myocardial LD protein) in a murine cDNA data base and showed the mRNA and protein to be highly enriched in the heart and also expressed at lower levels in the liver and adrenals. Upon subcellular fractionation, a substantial amount of MLDP was detected in the top fraction enriched with LDs. Furthermore, overexpressed MLDP tagged with green fluorescent protein accumulated at the surfaces of LDs and co-localized with perilipin and ADRP. Deletion analysis demonstrated the N-terminal region containing a PAT-1 domain and the following 33-mer domain to be required for targeting of MLDP to LDs. MLDP was found to be up-regulated at both mRNA and protein levels in the heart and liver by a selective ligand for PPARalpha, Wy14,643, but not in PPARalpha knock-out mice. MLDP expression was also increased upon fasting in parallel with ADRP. These results indicate that MLDP is a bona fide new PAT family member localized in LDs. Its expression depends on the physiological conditions and the action of PPARalpha.

Protection against fatty liver but normal adipogenesis in mice lacking adipose differentiation-related protein.

Adipose differentiation-related protein (ADFP; also known as ADRP or adipophilin), is a lipid droplet (LD) protein found in most cells and tissues. ADFP expression is strongly induced in cells with increased lipid load. We have inactivated the Adfp gene in mice to better understand its role in lipid accumulation. The Adfp-deficient mice have unaltered adipose differentiation or lipolysis in vitro or in vivo. Importantly, they display a 60% reduction in hepatic triglyceride (TG) and are resistant to diet-induced fatty liver. To determine the mechanism for the reduced hepatic TG content, we measured hepatic lipogenesis, very-low-density lipoprotein (VLDL) secretion, and lipid uptake and utilization, all of which parameters were shown to be similar between mutant and wild-type mice. The finding of similar VLDL output in the presence of a reduction in total TG in the Adfp-deficient liver is explained by the retention of TG in the microsomes where VLDL is assembled. Given that lipid droplets are thought to form from the outer leaflet of the microsomal membrane, the reduction of TG in the cytosol with concomitant accumulation of TG in the microsome of Adfp-/- cells suggests that ADFP may facilitate the formation of new LDs. In the absence of ADFP, impairment of LD formation is associated with the accumulation of microsomal TG but a reduction in TG in other subcellular compartments.

Long-Chain Fatty Acids Induce Lipid Droplet Formation in a Cultured Human Hepatocyte in a Manner Dependent of Acyl-CoA Synthetase

Lipid droplets (LDs) are intracellular storage sites of neutral lipids, which accumulate in fatty liver disease. Here, we investigated the effects of fatty acids and glucose on LD formation in a cultured human hepatocyte, HuH7, by adding them to culture media. Fatty acids with carbohydrate chains C12-C18 efficiently induced LDs, but those of C8 and C10 were ineffective. Glucose did not induce LD formation even in the presence of insulin. Oleic acid induced significant increases in cellular neutral lipids, and cell fractionation revealed that most of the newly synthesized neutral lipids were concentrated in LDs together with LD proteins. The LD formation was not abrogated by removal of medium glucose but was significantly inhibited by an ACSL inhibitor, triacsin C. These results demonstrate that long-chain fatty acids contribute to LD formation to a greater extent than glucose, possibly by being taken up into the cells, activated by ACSL, reconstituted into neutral lipids and then stored in LDs. Pregnenolone and lithium did not suppress oleic acid-dependent LD formation, despite previous reports of their ability to inhibit LD formation in macrophages and adipocytes suggesting differences among LD formations in these cells.

Fixation and permeabilization protocol is critical for the immunolabeling of lipid droplet proteins

The number of proteins known to be associated with lipid droplets (LDs) is increasing. However, the reported distribution of a given protein in the LDs was, in some cases, found not reproduced by other groups. We report here that the choice of the fixation and permeabilization method is important in order to observe LD proteins using immunofluorescence microscopy. Formaldehyde fixation followed by treatment with Triton X-100, one of the most frequently used protocols for the immunolabeling of cultured cells, was not appropriate to label adipocyte differentiation-related protein (ADRP), TIP47, or Rab18 in LDs. Formaldehyde fixation followed by treatment with digitonin or saponin, allowed the visualization of all these proteins in LDs. When cells were fixed with glutaraldehyde, permeabilization by Triton X-100 could also be used for ADRP. These observations suggest that LD proteins are likely to be solubilized by some detergents, and strong cross-linkage to the surrounding protein matrix or mild permeabilization is necessary for their retention on the LD surface.

Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body

The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42-44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak approximately 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.

Adipose Differentiation-Related Protein: A Gonadotropin- and Prostaglandin-Regulated Protein in Primate Periovulatory Follicles1

The midcycle LH surge stimulates a rise in follicular fluid prostaglandin E2 (PGE2), which is necessary for normal ovulation. To examine PGE2-regulated processes in primate follicles, monkey granulosa cells were cultured with hCG alone or with hCG and PGE2, and the resulting total RNA was subjected to microarray analysis. Twenty PGE2-regulated mRNAs were identified, and we selected a lipid droplet protein, adipose differentiation-related protein (ADRP), for further study. To determine whether hCG and PGE2 regulate ADRP expression in vivo, monkeys received gonadotropins to stimulate multiple follicular development. Human chorionic gonadotropin was then administered alone or with the PG synthesis inhibitor celecoxib, and follicular aspirates or whole ovaries were obtained at times that span the 40-h periovulatory interval. Administration of hCG increased granulosa cell ADRP mRNA and protein, with peak levels measured just before the expected time of ovulation. Treatment with hCG and celecoxib decreased granulosa cell ADRP mRNA levels compared with those of animals treated with hCG only. ADRP was detected by immunocytochemistry in many monkey tissues that synthesize prostaglandins but was not consistently expressed by steroidogenic tissues. Granulosa cells of periovulatory follicles immunostained for ADRP after, but not before, hCG administration; ADRP colocalized with large lipid droplets within the granulosa cell cytoplasm. These studies identify ADRP as a novel gonadotropin- and PGE2-regulated protein in the granulosa cells of primate periovulatory follicles. Because ADRP facilitates arachidonic acid uptake in non-ovarian cells, ADRP-associated lipid droplets may enhance arachidonic acid uptake by granulosa cells to provide a precursor for periovulatory prostaglandin production.

PPARγ2 regulates lipogenesis and lipid accumulation in steatotic hepatocytes

Peroxisome proliferator-activated receptor-gamma (PPARgamma) is considered to be one of the master regulators of adipocyte differentiation. PPARgamma2 is abundantly expressed in mature adipocytes and is elevated in the livers of animals that develop fatty livers. The aim of this study was to determine the ability of PPARgamma2 to induce lipid accumulation in hepatocytes and to delineate molecular mechanisms driving this process. The hepatic cell line AML-12 was used to generate a cell line stably expressing PPARgamma2. Oil Red O staining revealed that PPARgamma2 induces lipid accumulation in hepatocytes. This phenotype is accompanied by a selective upregulation of several adipogenic and lipogenic genes including adipose differentiation-related protein (ADRP), adipocyte fatty acid-binding protein 4, sterol regulatory element-binding protein-1 (SREBP-1), fatty acid synthase (FAS), and acetyl-CoA carboxylase, genes whose expression levels are known to increase in steatotic livers of ob/ob mice. Furthermore, the PPARgamma2-regulated induction of both SREBP-1 and FAS parallels an increase in de novo triacylglycerol synthesis in hepatocytes. Triacylglycerol synthesis and lipid accumulation are further enhanced by culturing hepatocytes with troglitazone in the absence of exogenous lipids. These results correspond with an increase in the lipid droplet protein, ADRP, and the data demonstrate that ADRP functions to coat lipid droplets in hepatocytes as observed by confocal microscopy. Taken together, these observations propose a role for PPARgamma2 as an inducer of steatosis in hepatocytes and suggest that this phenomenon occurs through an induction of pathways regulating de novo lipid synthesis.

Metabolic Adaptations in the Absence of Perilipin: INCREASED β-OXIDATION AND DECREASED HEPATIC GLUCOSE PRODUCTION ASSOCIATED WITH PERIPHERAL INSULIN RESISTANCE BUT NORMAL GLUCOSE TOLERANCE IN PERILIPIN-NULL MICE

Targeted disruption of the lipid droplet protein, perilipin, in mice leads to constitutional lipolysis associated with marked reduction in white adipose tissue as a result of unbridled lipolysis. To investigate the metabolic adaptations in response to the constitutive lipolysis, we studied perilipin-null (plin(-/-)) mice in terms of their fatty acid oxidation and glycerol and glucose metabolism homeostasis by using dynamic biochemical testing and clamp and tracer infusion methods. plin(-/-) mice showed increased beta-oxidation in muscle, liver, and adipose tissue resulting from a coordinated regulation of the enzymes and proteins involved in beta-oxidation. The increased beta-oxidation helped remove the extra free fatty acids created by the constitutive lipolysis. An increase in the expression of the transcripts for uncoupling proteins-2 and -3 also accompanied this increase in fatty acid oxidation. Adult plin(-/-) mice had normal plasma glucose but a reduced basal hepatic glucose production (46% that of plin(+/+)). Insulin infusion during low dose hyperinsulinemic-euglycemic clamp further lowered the glucose production in plin(-/-) mice, but plin(-/-) mice also showed a 36% decrease (p < 0.007) in glucose disposal rate during the low dose insulin clamp, indicating peripheral insulin resistance. However, compared with plin(+/+) mice, 14-week-old plin(-/-) mice showed no significant difference in glucose disposal rate during the high dose hyperinsulinemic clamp, whereas 42-week-old plin(-/-) mice displayed significant insulin resistance on high dose hyperinsulinemic clamp. Despite increasing insulin resistance with age, plin(-/-) mice at different ages maintained a normal glucose response during an intraperitoneal glucose tolerance curve, being compensated by the increased beta-oxidation and reduced hepatic glucose production. These experiments uncover the metabolic adaptations associated with the constitutional lipolysis in plin(-/-) mice that allowed the mice to continue to exhibit normal glucose tolerance in the presence of peripheral insulin resistance.

Immunocytochemical studies on lipid droplet-surface proteins in adrenal cells.

Perilipin and ADRP, located on the surface of intracellular lipid droplets, are proposed to be involved in adipocyte lipid metabolism. The aim of the present study was to investigate the effect of PKA and PKC activities on the distribution of perilipin and ADRP in primary cultured adrenal cells, and the role of ERK in PMA- and calphostin C-induced steroidogenesis. Immunofluorescence staining indicated that in addition to p160, a capsular protein of steroidogenic lipid droplets, perilipin and ADRP were localized on the lipid droplet surface. Stimuli such as activation of PKA by db cAMP or inhibition of PKC by calphostin C, which increase corticosterone synthesis in various magnitudes, caused detachment of p160 and perilipin, but not ADRP, from the lipid droplet surface. Activation of PKC by PMA induced increase in corticosterone synthesis, however, it did not affect the distribution of perilipin, p160, or ADRP on the lipid droplet surface, suggesting the presence of mechanisms for promoting sterodiogensis other than causing detachment of lipid droplet surface proteins. We further demonstrated that ERK pathway was involved in PMA-induced steroidogenesis, since PD98059, specific inhibitor of MEK, blocked the increases in steroidogenesis and phosphorylation of ERK caused by PMA, but not by cAMP-PKA. These data indicate that p160, perilipin, and ADRP were all located on the lipid droplet surface in rat adrenal cells. On the basis of its non-responsiveness to lipolytic stimulation, ADRP may be a structural protein of the lipid droplet surface, whereas their immediate response to lipolytic stimuli suggest that perilipin and p160 are functional proteins. PKC regulates adrenal steroidogenesis through ERK cascade, whereas PKA pathway does not involve ERK.

TIP47 Associates with Lipid Droplets

Most mammalian cells package neutral lipids into droplets that are surrounded by a monolayer of phospholipids and a specific set of proteins including the adipose differentiation-related protein (ADRP; also called adipophilin), which is found in a wide array of cell types, and the perilipins, which are restricted to adipocytes and steroidogenic cells. TIP47 was initially identified in a yeast two-hybrid screen for proteins that interact with the cytoplasmic tail of the mannose 6-phosphate receptor, yet its sequence is highly similar to the lipid droplet protein, ADRP, and more distantly related to perilipins. Hence, we hypothesized that TIP47 might be associated with lipid droplets. In HeLa cells grown in standard low lipid-containing culture media, immunofluorescence microscopy revealed that the cells had few lipid droplets; however, TIP47 and ADRP were found on the surfaces of the small lipid droplets present. When the cells were grown in media supplemented with physiological levels of fatty acids, the amount of neutral lipid stored in lipid droplets increased dramatically, as did the staining of TIP47 and ADRP surrounding these droplets. TIP47 was found primarily in the cytosolic fractions of HeLa cells and murine MA10 Leydig cells grown in low lipid-containing culture medium, while ADRP was undetectable in these fractionated cell homogenates. When HeLa and MA10 Leydig cells were lipid-loaded, significant levels of ADRP were found in the floating lipid droplet fractions and TIP47 levels remained constant, but the distribution of a significant portion of TIP47 shifted from the cytosolic fractions to the lipid droplet fractions. Thus, we conclude that TIP47 associates with nascent lipid droplets and can be classified as a lipid droplet-associated protein.

Sterol Carrier Protein-2 Expression Modulates Protein and Lipid Composition of Lipid Droplets

Despite the critical role lipid droplets play in maintaining energy reserves and lipid stores for the cell, little is known about the regulation of the lipid or protein components within the lipid droplet. Although immunofluorescence of intact cells as well as Western analysis of isolated lipid droplets revealed that sterol carrier protein-2 (SCP-2) was not associated with lipid droplets, SCP-2 expression significantly altered the structure of the lipid droplet. First, the targeting of fatty acid and cholesterol to the lipid droplets was significantly decreased. Second, the content of several proteins important for lipid droplet function was differentially increased (perilipin A), reduced severalfold (adipose differentiation-related protein (ADRP), vimentin), or almost completely eliminated (hormone-sensitive lipase and proteins >93 kDa) in the isolated lipid droplet. Third, the distribution of lipids within the lipid droplets was significantly altered. Double labeling of cells with 12-(N-methyl)-N-[(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-octadecanoic acid (NBD-stearic acid) and antisera to ADRP showed that 70, 24, and 13% of lipid droplets contained ADRP, NBD-stearic acid, or both, respectively. SCP-2 expression decreased the level of ADRP in the lipid droplet but increased the proportion wherein ADRP and NBD-stearic acid colocalized by 3-fold. SCP-2 expression also decreased the lipid droplet fatty acid and cholesterol mass (nmol/mg protein) by 5.2- and 6.6-fold, respectively. Finally, SCP-2 expression selectively altered the pattern of esterified fatty acids in favor of polyunsaturated fatty acids within the lipid droplet. Displacement studies showed differential binding affinity of ADRP for cholesterol and fatty acids. These data suggested that SCP-2 and ADRP play a significant role in regulating fatty acid and cholesterol targeting to lipid droplets as well as in determining their lipid and protein components.

Sequence motifs required for lipid droplet association and protein stability are unique to the hepatitis C virus core protein.

From analysis of the primary sequence of the hepatitis C virus (HCV) core protein, we have identified three separable regions based on hydrophobicity and clustering of basic amino acids within the protein. Comparison with capsid proteins of related pesti- and flaviviruses suggested that HCV core has a unique central domain (domain 2). Previous findings have revealed that core protein can associate with lipid droplets which are intracellular storage sites for triacylglycerols and cholesterol esters. Confocal analysis of variant forms lacking regions of core indicated that most residues within the unique region are necessary for association of the protein with lipid droplets. A segment within domain 2 (from residues 125 to 144) also was required for stability of the protein and a polypeptide lacking these sequences was degraded apparently by the proteasome. In cells depleted of lipid droplets, core protein remained located in the cytoplasm. Moreover, cleavage of the protein at the maturation site and stability were not affected by inability to bind to lipid droplets.

Morphology of the release of vitamin A-containing lipid droplets by perisinusoidal lipocytes in the rat liver.

Vitamin A-containing lipid droplets in perisinsoidal lipocytes (or fat-storing cells) of rat liver were found to be exocytotically released from the cells in the form of a 'lipid droplet-protein complex' following intraportal injection of retinol (17, 33, 67 or 100 micrograms). Intraportal injection of retinol produced varied numbers and sizes of vacuoles with a limiting membrane in perisinusoidal lipocytes. The substance within the vacuoles exhibited a meshwork-like configuration in sections from slices incubated in a medium for revealing acid phosphatase activity or the corresponding control medium, and was immunoreactive to retinol-binding protein and proteinaceous in nature. The occurrence and number of the vacuoles depended on the dosage of injected retinol, being greatest at a dosage of 100 micrograms of retinol and becoming progressively less at dosages of 67, 33 and 17 micrograms. The vacuoles appeared to be formed by vacuolization of cisternae of the rough endoplasmic reticulum. Little or no esterase activity was found in lipid droplets in perisinusoidal lipocytes before the intraportal injection of retinol, but after the injection lipid droplets which had fused with the vacuoles became strongly positive for this enzyme activity. This suggests that hydrolysis of retinyl esters may occur in the process of complex formation in perisinusoidal lipocytes.