Temporal and spatial assembly of lipid droplet-associated proteins in 3T3-L1 preadipocytes

This study aimed to investigate the relationship between newly formed lipid droplets and lipid droplet surface proteins, including perilipin, adipose differentiation related protein (ADRP), and p200 kDa protein (p200) in 3T3-L1 preadipocytes, during lipogenesis. Sterol ester was used to induce nascent lipid droplets in 3T3-L1 preadipocytes and the sequence of lipids and lipid droplet surface proteins was studied using a combination of immunohistochemistry and Nile red staining/Oil red O. We demonstrated that, although most growing lipid droplets appeared to have a lipid core surrounded by a fluorescent rim of ADRP, perilipin, and p200, tiny protein aggregates of ADRP, perilipin, or p200 could also be found to occur in the absence of lipid accumulation. In addition, ADRP associated with nascent lipid droplets prior to that of perilipin or p200. We provide evidence that lipid droplet surface proteins, especially ADRP and perilipin, are important in serving as a nucleation center for the assembly of lipid to form nascent lipid droplets.     

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.     

ADRP stimulates lipid accumulation and lipid droplet formation in murine fibroblasts

Adipose differentiation-related protein (ADRP) is a lipid droplet-associated protein that is expressed early during adipose differentiation. The present study was undertaken to reveal the role of ADRP in adipose differentiation. In murine fibroblasts infected with green fluorescent protein (GFP)-ADRP fusion protein expression adenovirus vector, confocal microscopic analysis showed the number and size of lipid droplets apparently increased comparing with those of control cells. Overexpressed GFP-ADRP were mainly located at the surface of lipid droplets and appeared to be "ring-shaped." Triacylglycerol content was also significantly (P < 0.001) increased in GFP-ADRP-overexpressed cells compared with control cells. ADRP-induced lipid accumulation did not depend on adipocyte-specific gene induction, such as peroxisome proliferator-activated receptor-gamma, lipoprotein lipase, or other lipogenic genes, including acyl-CoA synthetase, fatty acid-binding protein, and fatty acid transporter. In conclusion, ADRP stimulated lipid accumulation and lipid droplet formation without induction of other adipocyte-specific genes or other lipogenic genes in murine fibroblasts. The detailed molecular mechanisms of ADRP on lipid accumulation remain to be elucidated.     

Live cell analysis and targeting of the lipid droplet-binding adipocyte differentiation-related protein

Neutral lipid is stored in spherical organelles called lipid droplets that are bounded by a coat of proteins. The protein that is most frequently found at the surface of lipid droplets is adipocyte differentiation-related protein (ADRP). In this study, we demonstrate that fusion of either the human or mouse ADRP coding sequences to green fluorescent protein (GFP) does not disrupt the ability of the protein to associate with lipid droplets. Using this system to identify targeting elements, discontinuous segments within the coding region were required for directing ADRP to lipid droplets. GFP-tagged protein was employed also to examine the behavior of lipid droplets in live cells. Time lapse microscopy demonstrated that in HuH-7 cells, which are derived from a human hepatoma, a small number of lipid droplets could move rapidly, indicating transient association with intracellular transport pathways. Most lipid droplets did not show such movement but oscillated within a confined area; these droplets were in close association with the endoplasmic reticulum membrane and moved in concert with the endoplasmic reticulum. Fluorescence recovery analysis of GFP-tagged ADRP in live cells revealed that surface proteins do not rapidly diffuse between lipid droplets, even in conditions where they are closely packed. This system provides new insights into the properties of lipid droplets and their interaction with cellular processes.     

Hepatitis C Virus Increases Occludin Expression via the Upregulation of Adipose Differentiation-Related Protein

The hepatitis C virus (HCV) life cycle is closely associated with lipid metabolism. In particular, HCV assembly initiates at the surface of lipid droplets. To further understand the role of lipid droplets in HCV life cycle, we assessed the relationship between HCV and the adipose differentiation-related protein (ADRP), a lipid droplet-associated protein. Different steps of HCV life cycle were assessed in HCV-infected human Huh-7 hepatoma cells overexpressing ADRP upon transduction with a lentiviral vector. HCV infection increased ADRP mRNA and protein expression levels by 2- and 1.5-fold, respectively. The overexpression of ADRP led to an increase of (i) the surface of lipid droplets, (ii) the total cellular neutral lipid content (2.5- and 5-fold increase of triglycerides and cholesterol esters, respectively), (iii) the cellular free cholesterol level (5-fold) and (iv) the HCV particle production and infectivity (by 2- and 3.5-fold, respectively). The investigation of different steps of the HCV life cycle indicated that the ADRP overexpression, while not affecting the viral replication, promoted both virion egress and entry (~12-fold), the latter possibly via an increase of its receptor occludin. Moreover, HCV infection induces an increase of both ADRP and occludin expression. In HCV infected cells, the occludin upregulation was fully prevented by the ADRP silencing, suggesting a specific, ADRP-dependent mechanism. Finally, in HCV-infected human livers, occludin and ADRP mRNA expression levels correlated with each other. Alltogether, these findings show that HCV induces ADRP, which in turns appears to confer a favorable environment to viral spread.     

Magnolol stimulates lipolysis in lipid-laden RAW 264.7 macrophages

This study investigated the effect of magnolol, a compound isolated from Magnolia officinalis, on lipolysis in lipid-laden RAW 264.7 macrophages. Treatment of macrophages with magnolol led to dissolution of lipid droplets. This phenomenon was accompanied by a dose-dependent release of glycerol and cholesterol and a concomitant reduction in intracellular levels of glycerol and cholesterol. Furthermore, adipose differentiation-related protein (ADRP), a lipid droplet-associated protein, was down-regulated by magnolol in a dose- and time-dependent manner by Western blot analysis. Immunofluorescence studies also showed that ADRP became detached from the surface of lipid droplets after magnolol treatment. The lipolytic effect of magnolol was not mediated through the cAMP-protein kinase A (PKA) system, an authentic lipolytic pathway for macrophages, since magnolol did not induce an increase of intracellular cAMP levels, and pretreatment with either of PKA inhibitors, PKI and KT5720, did not abrogate the lipolytic response to magnolol. We conclude that magnolol induce-lipolysis of lipid-laden macrophages by down-regulation of ADRP expression and detachment of ADRP from the lipid droplet surface by a cAMP-independent mechanism. Lipolysis of lipid-laden macrophages may occur when the amount of ADRP on the surface of lipid droplets is not enough to stabilize the lipid droplets.     

Adipocyte differentiation-related protein reduces the lipid droplet association of adipose triglyceride lipase and slows triacylglycerol turnover

Although neutral lipid storage droplets are ubiquitous in eukaryotic cells, very little is known about how their synthesis and turnover are controlled. Adipocyte differentiation-related protein (ADRP; also known as adipophilin) is found on the surface of lipid droplets in most mammalian cell types. To learn how ADRP affects lipid storage, we stably expressed the protein in human embryonic kidney 293 (HEK 293) cells, which express little endogenous ADRP. As expected, ADRP was targeted to the surface of lipid droplets and caused an increase in triacylglycerol (TAG) mass under both basal and oleate-supplemented conditions. At least part of the increased mass resulted from a 50% decrease in the rate of TAG hydrolysis in ADRP-expressing cells. Furthermore, ADRP expression increased the fraction of total cellular TAG that was stored in lipid droplets. ADRP expression induced a striking decrease in the association of adipose triglyceride lipase (ATGL) and mannose-6-phosphate receptor tail-interacting protein of 47 kDa with lipid droplets and also decreased the lipid droplet association of several other unknown proteins. Transient expression of ADRP in two other cell lines also reduced the lipid droplet association of catalytically inactive ATGL. We conclude that the reduced lipid droplet association of ATGL and/or other lipases may explain the decrease in TAG turnover observed in ADRP-expressing HEK 293 cells.     

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.     

Post-translational regulation of adipose differentiation-related protein by the ubiquitin/proteasome pathway

Adipose differentiation-related protein (ADRP) is localized to lipid droplets in most mammalian cells. ADRP, proposed to regulate fatty acid mobilization and lipid droplet formation, is linked to lipid accumulation in foam cells of human atherosclerotic lesions. In this report, we show that ADRP protein accumulates in Chinese hamster ovary fibroblastic cells cultured in the presence of oleic acid but is destabilized when fatty acid sources are removed from culture serum. The latter effect was blocked by the proteasome inhibitor MG132, whereas inhibitors of other proteolytic processes were ineffective. Pulse-chase experiments confirmed that ADRP degradation is inhibited by MG132. Conditions that stimulate ADRP degradation also promoted the covalent modification of ADRP by ubiquitin, whereas the addition of oleic acid to culture media, which promotes triacylglycerol deposition, blunted the appearance of ubiquitinated-ADRP. Treatment with MG132 increased the levels of ADRP associated with lipid droplets, as well as throughout the cytosol. Finally, we demonstrate that the disappearance of ADRP protein after the onset of perilipin expression during adipocyte differentiation is due to degradation by proteasomes Thus, proteolytic degradation of ADRP mediated through the ubiquitin/proteasome pathway appears to be a major mode for the post-translational regulation of ADRP.     

ADRP/adipophilin is degraded through the proteasome-dependent pathway during regression of lipid-storing cells

Adipose differentiation-related protein (ADRP) is a major protein associated with lipid droplets in various types of cells, including macrophage-derived foam cells and liver cells. However, the role of ADRP in the processes of formation and regression of these cells is not understood. When J774 murine macrophages were incubated with either VLDL or oleic acid, their content of both ADRP and triacylglycerol (TG) increased 3- to 4-fold. Induction of ADRP during TG accumulation was also observed in oleic acid-treated HuH-7 human liver cells. Addition of triacsin C, a potent inhibitor of acyl-CoA synthase, for 6 h decreased the amount of TG in VLDL-induced foam cells and oleic acid-treated liver cells; it decreased the amount of ADRP protein in parallel, indicating the amount of ADRP reduced during regression of the lipid-storing cells. Addition of a proteasome inhibitor during triacsin C treatment abolished the ADRP decrease and accumulated polyubiquitinated ADRP. In addition, the proteasome inhibitor reversed not only the degradation of ADRP but also TG reduction by triacsin C. These results suggest that cellular amounts of ADRP and TG regulate each other and that the ubiquitin-proteasome system is involved in degradation of ADRP during regression of lipid-storing cells.     

Adipocyte differentiation-related protein is induced by LRP1-mediated aggregated LDL internalization in human vascular smooth muscle cells and macrophages

Aggregated LDL (agLDL) is internalized by LDL receptor-related protein (LRP1) in vascular smooth muscle cells (VSMCs) and human monocyte-derived macrophages (HMDMs). AgLDL is, therefore, a potent inducer of massive intracellular cholesteryl ester accumulation in lipid droplets. The adipocyte differentiation-related protein (ADRP) has been found on the surface of lipid droplets. The objectives of this work were to analyze whether agLDL uptake modulates ADRP expression levels and whether the effect of agLDL internalization on ADRP expression depends on LRP1 in human VSMCs and HMDMs. AgLDL strongly upregulates ADRP mRNA (real-time PCR) and protein expression (Western blot) in human VSMCs (mRNA: by 3.06-fold; protein: 8.58-fold) and HMDMs (mRNA: by 3.5-fold; protein: by 3.71-fold). Treatment of VSMCs and HMDMs with small anti-LRP1-interfering RNA (siRNA-LRP1) leads to specific inhibition of LRP1 expression. siRNA-LRP1 treatment significantly reduced agLDL-induced ADRP overexpression in HMDMs (by 69%) and in VSMCs (by 53%). Immunohystochemical studies evidence a colocolocalization between ADRP/macrophages and ADRP/VSMCs in advanced lipid-enriched atherosclerotic plaques. These results demonstrate that agLDL-LRP1 engagement induces ADRP overexpression in both HMDMs and human VSMCs and that ADRP is highly expressed in advanced lipid-enriched human atherosclerotic plaques. Therefore, LRP1-mediated agLDL uptake might play a pivotal role in vascular foam cell formation.     

脂肪分化相关蛋白的研究进展

细胞内脂滴是一种代谢活跃的细胞器,脂滴表面蛋白在脂滴的代谢调节中起到了重要作用。ADRP是一种重要的脂滴表面蛋白,在机体组织和细胞内广泛表达。脂肪肝、动脉粥样硬化、糖尿病等均伴随脂质的异常蓄积,近年来的研究表明ADRP参与这些疾病的发生发展。本文就ADRP在各组织和器官正常的生理功能以及对疾病状态的调控加以综述。     

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.     

Up-regulation of ADRP in fatty liver in human and liver steatosis in mice fed with high fat diet

The present study was performed to examine a role of adipose differentiation-related protein (ADRP) in the process of liver steatosis. Immunohistochemical findings indicated that ADRP expression is increased in the hepatocytes in patients with fatty liver when compared with normal liver. ADRP expression is localized in the surface of lipid droplets in the hepatocytes. Increased expression of ADRP mRNA and protein was similarly observed in fatty liver in ob/ob mice and the liver steatosis induced by high fat diet in mice. The up-regulation of ADRP mRNA and protein in the liver by high fat diet was identified in the surface of lipid droplets in a time-dependent manner. Recent studies demonstrated that up-regulation of PPARgamma in the hepatocytes is deeply involved in liver steatosis. To clarify whether ADRP expression is increased by PPARgamma activation in hepatocytes, we examined the effect of a PPARgamma ligand, troglitazone, on ADRP mRNA expression in HepG2 cells. ADRP mRNA expression was increased by troglitazone in dose- and time-dependent manners. All these results suggest that ADRP is up-regulated in liver steatosis in human and mice, and that high fat diet increases expression of ADRP through PPARgamma activation, followed by induction of liver steatosis.     

Glucagon regulates intracellular distribution of adipose differentiation-related protein during triacylglycerol accumulation in the liver

Cellular lipid droplets (LD) are organelles involved in cellular lipid metabolism. When liver cellular components were fractionated using sucrose density gradient centrifugation, adipose differentiation-related protein (ADRP) was distributed in both the top and bottom fractions, which correspond to the LD and membranous fractions, respectively, in the mouse liver under normal feeding conditions. After overnight fasting, triacylglycerol and ADRP increased nearly 2.5-fold in the mouse liver, and a portion appeared in the intermediate-density LD (iLD) fractions. ADRP in the iLD fractions was also increased in a mouse nonalcoholic steatohepatitis model induced by methione/choline-deficient diet. When HuH-7 human hepatoma cells were incubated with oleic acid for 24 h, the amount of ADRP increased, and it was distributed in both the LD and membrane fractions. However, ADRP appeared in the iLD fractions upon treatment of HuH-7 cells with glucagon. This behavior of ADRP was cAMP-dependent, as the ADRP-positive iLD fractions were induced by dibutylyl cAMP and were blocked by protein kinase A inhibitors. A portion of ADRP colocalized microscopically with calnexin, which is present in the iLD fractions, by treatment of HuH-7 cells or human primary hepatocytes with oleic acid and glucagon, but not by treatment with oleic acid alone. Glucagon has a role in the reorganization of endoplasmic reticulum membranes to generate ADRP-associated lipid-poor particles in hepatic cells, which is related to LD formation during lipid storage.     

Adipose differentiation related protein induces lipid accumulation and lipid droplet formation in hepatic stellate cells

Summary The function of adipose differentiation-related protein (ADRP) is known to be the uptake of long-chain fatty acids and formation of lipid droplets in lipid-accumulating cells. We hypothesized that ADRP might stimulate activated hepatic stellate cells (HSCs) to accumulate lipids, resulting in their transition to the quiescent state. In this study, cultured HSCs in fifth passages isolated from rat were infected by adenovirus vector expressing ADRP (Ad.GFP-ADRP), and morphologic and functional changes were evaluated in comparison with control HSCs infected by recombinant adenovirus-expressing β-galactosidase (Ad. LacZ). In Ad. GFP-ADRP-infected cells only, many tiny lipid droplets were apparent in the cytoplasm, while the outline of the cells was not changed. The ADRP was detected around the lipid droplets. In HSCs with intracellular actin filaments, the staining pattern of the filaments before and after infection with Ad.GFP-ADRP or Ad.LacZ did not differ. The cell proliferation rate was not influenced by infection with Ad.LacZ or Ad.GFP-ADRP. Type I collagen secretion from cells overexpressing ADRP was not significantly different from that of Ad.LacZ-infected cells. In our in vitro study, ADRP overexpression induced the formation of cytoplasmic lipid droplets in activated HSCs but could not convert other characteristics of the activated form into those of the quiescent form.     

Accumulation of caveolin in the endoplasmic reticulum redirects the protein to lipid storage droplets

Caveolin-1 is normally localized in plasma membrane caveolae and the Golgi apparatus in mammalian cells. We found three treatments that redirected the protein to lipid storage droplets, identified by staining with the lipophilic dye Nile red and the marker protein ADRP. Caveolin-1 was targeted to the droplets when linked to the ER-retrieval sequence, KKSL, generating Cav-KKSL. Cav-DeltaN2, an internal deletion mutant, also accumulated in the droplets, as well as in a Golgi-like structure. Third, incubation of cells with brefeldin A caused caveolin-1 to accumulate in the droplets. This localization persisted after drug washout, showing that caveolin-1 was transported out of the droplets slowly or not at all. Some overexpressed caveolin-2 was also present in lipid droplets. Experimental reduction of cellular cholesteryl ester by 80% did not prevent targeting of Cav-KKSL to the droplets. Cav-KKSL expression did not grossly alter cellular triacylglyceride or cholesteryl levels, although droplet morphology was affected in some cells. These data suggest that accumulation of caveolin-1 to unusually high levels in the ER causes targeting to lipid droplets, and that mechanisms must exist to ensure the rapid exit of newly synthesized caveolin-1 from the ER to avoid this fate.     

Dynamics of lipid droplet-associated proteins during hormonally stimulated lipolysis in engineered adipocytes: stabilization and lipid droplet binding of adipocyte differentiation-related protein/adipophilin

In mature adipocytes, triglyceride is stored within lipid droplets, which are coated with the protein perilipin, which functions to regulate lipolysis by controlling lipase access to the droplet in a hormone-regulatable fashion. Adipocyte differentiation-related protein (ADRP) is a widely expressed lipid droplet binding protein that is coexpressed with perilipin in differentiating fat cells but is minimally present in fully differentiated cultured adipocytes. We find that fibroblasts ectopically expressing C/EBPalpha (NIH-C/EBPalpha cells) differentiate into mature adipocytes that simultaneously express perilipin and ADRP. In response to isoproterenol, perilipin is hyperphosphorylated, lipolysis is enhanced, and subsequently, ADRP expression increases coincident with it surrounding intracellular lipid droplets. In the absence of lipolytic stimulation, inhibition of proteasomal activity with MG-132 increased ADRP levels to those of cells treated with 10 mum isoproterenol, but ADRP does not surround the lipid droplet in the absence of lipolytic stimulation. We overexpressed a perilipin A construct in NIH-C/EBPalpha cells where the six serine residues known to be phosphorylated by protein kinase A were changed to alanine (Peri A Delta1-6). These cells show no increase in ADRP expression in response to isoproterenol. We propose that ADRP can replace perilipin on existing lipid droplets or those newly formed as a result of fatty acid reesterification, under dynamic conditions of hormonally stimulated lipolysis, thus preserving lipid droplet morphology/structure.