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J. Biol. Chem., Vol. 281, Issue 45, 34341-34348, November 10, 2006
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1
2
From the
Geriatric Research, Education and Clinical Center, Baltimore Veterans Affairs Health Care Center, Department of Medicine, School of Medicine, University of Maryland, Baltimore, Maryland 21201, the Laboratory of Cellular and Developmental Biology, NIDDK, National Institutes of Health, Bethesda, Maryland 20892-8028, and the ¶Departments of Molecular and Cellular Biology and Medicine, Baylor College of Medicine, Houston, Texas 77030
Received for publication, March 16, 2006 , and in revised form, September 11, 2006.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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The mammalian PAT family includes five members: perilipin, ADFP,3 tail interacting protein of 47 kDa (Tip47), S3-12, and PAT-1 (9). The PAT proteins are defined by primary sequence homology and are well conserved within the family and across species (10). Recent proteomic studies revealed heterogeneity and tissue-specific differences in droplet-associated proteins (4-8). PAT protein distribution is clearly tissue-dependent. Perilipin and S3-12 are confined to adipose and steroidogenic tissues, while ADFP and Tip47 are ubiquitously distributed (11-14). To date, a functional role regulating lipolysis has been shown for perilipin, the principal lipid droplet protein in adipose cells, mainly from studies of the perilipin null mouse that exhibits a lean phenotype (15, 16), but little is known about the function of the other PAT proteins, including the proteins associated with lipid droplets in non-adipogenic tissues, such as ADFP and Tip47.
ADFP has long been identified for association with the lipid droplet surface (13, 17). Discovered by Serrero and co-workers (18) as an early gene of adipocyte differentiation, the ADFP protein occurs only transiently in adipocytes. ADFP is present at the surface of nascent lipid droplets in early stages of adipocyte differentiation but reduced dramatically after the onset of perilipin expression, whereas ADFP mRNA remains elevated. In mature adipocytes ADFP is replaced by perilipin as the major lipid droplet protein. Thus it is not surprising that adipocyte lipid droplets in perilipin null mice are coated with ADFP. However, while adipocyte differentiation in perilipin null mice is not compromised (15, 16), the protein kinase A regulation of lipolysis is lost (16). Despite its strong primary sequence homology to perilipin, ADFP is not phosphorylated by protein protein kinase A and ADFP does not supplant the protein kinase A-regulated function of perilipin (16, 19).
Despite numerous attempts, we have failed to detect any effect of overexpression of ADFP mRNA on lipid metabolism in various types of cells.4 On the other hand, we have reported that protection of ADFP from proteosomal degradation with the inhibitor, MG-132, is accompanied by a substantial increase (170%) in lipid content compared with control cells untreated with MG-132, suggesting that ADFP, like perilipin, protects neutral lipids from degradation by lipases (20, 21).
A recent addition to the pool of known lipid droplet surface proteins is Tip47. This member of the PAT family was first identified as a possible cargo protein involved in the trafficking of the mannose-6-phosphate receptor and mainly studied as such (22). Tip47 is also known as placental protein pp17b (23) and closely related in primary sequence to ADFP (40%), prompting studies looking for its subcellular localization on lipid droplets (10, 14). Indeed, Tip47 was found to have increased association with the fat cake fraction when HeLa cells were incubated with oleic acid. Subsequently, its localization at the lipid droplet surface was confirmed in various types of cells (23, 24). Tip47, like ADFP, is widely distributed among non-adipogenic tissues but unlike both perilipin and ADFP; it is present in both lipid droplet and cytosolic compartments. This unique property of solubility among the PAT proteins permitted the solution crystal structure of the COOH-terminal portion of Tip47 (25). ADFP null mice exhibit few phenotypic alterations, except for mild resistance to high fat diet-induced fatty liver (26). We report herein that lipid droplets in fibroblastic cells derived from ADFP null mice are coated with Tip47. Using these ADFP null MEF cells, we show that knockdown of Tip47 in these cells with siRNA decreased lipid storage, demonstrating the critical role of PAT proteins in the packaging and storage of neutral lipids in non-adipose cells.
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EXPERIMENTAL PROCEDURES |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Cell Lines and Cell Culture Conditions—wt and ADFP null fibroblastic cells were studied in parallel. For siRNA experiments, cells were plated in 6- or 24-multiwell dishes (Costar) and later used for metabolic studies, immunoblot and protein assays. For immunocytochemical studies, cells were plated on Petri dishes containing glass bottoms (MatTek Corp.) or 4-8-well chamber slides (Labtech). For isolation of lipid droplets by subcellular fractionation, cells were grown in 100 x 20-mm dishes (Costar). CHO-K1 cells were cultured in Ham's F-12 medium supplemented with 10% fetal calf serum (HyClone), 100 units/ml penicillin, 100 µg/ml streptomycin, and 2 mM glutamine (Biofluids, Inc.). Clones of CHO-K1 cells overexpressing Tip47-GFP fusion protein or ADFP-GFP fusion protein were selected in the presence of 600 µg/ml G418. For cellular lipolysis or lipogenesis studies, cells were plated in 24-multiwell dishes (Startdest) at a concentration of 1 x 104 cells/well. All cells were cultured in a 5% CO2 atmosphere at 37 °C.
Oligonucleotides—siRNAs were designed and purchased from Qiagen (HP guaranteed siRNA), and transfection methods for fibroblastic cell lines were used according to the manufacturer's instructions. The ability to down-regulate Tip47 was tested in both ADFP null and wt cells. Positive siRNAs for mitogen-activated protein kinase-1 and a rhodamine fluorescent oligonucleotide (Qiagen) were used to help establish efficient conditions for transfection.
siRNA Construction and Transfection—The siRNAs directed against mouse Tip47 cargo protein were designed by Qiagen. Cells were plated in antibiotic-free Dulbecco's modified Eagle's medium supplemented with 2% fetal calf serum at a concentration of 1 x 104 cells per well in a 24-multiwell dish. During the next 2 days, cells were transfected with siRNAs (20 nM) using RNAiFect (Qiagen) according to the manufacturer's instructions. Among six siRNAs for our target gene, the sequences specific for Tip47 cargo protein sens (5'-AACAGCACAGAGAAUGAGGAG-3') and sens (5'GAAUGAGACAUGUGUUUAA-3') and were selected based upon their potency to inhibit target gene expression. Equal amounts of two positive siRNAs were used. An ineffective siRNA was used as a negative control, sens (5'-GCGUGUCCAAUCAGUCAU-3'). For the lipogenesis experiments, wt and ADFP cells were transfected using Hyperfect (Qiagen) according to the manufacturer's instructions, cells were plated at a concentration of 1 x 104 cells per well with Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum in a 24-multiwell dish, and transfection with siRNA was performed the next day. In all experiments, cells were used for Western blotting and imunocytochemistry on day 5 after plating unless otherwise stated, since preliminary experiments have shown that day 5 was optimal for Tip47 protein down-regulation (effect of Tip47 siRNA measured by Western blotting was decreased after day 7 post-plating).
Full-length Tip47 and ADFP cDNA constructs were fused in-frame with the 3' end of eGFP in pEGFP-C2 (Clontech) as described previously (10). CHO cells were transfected with Lipofectamine, using either ADFP-GFP or Tip47-GFP fusion protein constructs. Clonal cell lines were established as described previously (19). Briefly, immediately following infection, CHO cells were placed under selection in 600 µg/ml G418, and cells after 8 days in selection medium were re-plated in 96-multiwells at a concentration of 2 cells/ml. Positive "green" clonal cells were identified by microscopy and further expanded.
Fat Cake Preparation—Cells were washed twice with phosphate-buffered saline, pH 7.4 (PBS), scraped into PBS, and pelleted by low speed centrifugation. Lipid droplet isolation was performed as described (14).
Antibodies and Fluorescent Dyes—Antibodies used include the following: anti-Tip47, which was raised in rabbits against the COOH-terminal 
200 amino acids of murine Tip47, and anti-ADFP, which was raised in rabbits against a peptide comprising the 26 amino-terminal amino acids of murine ADFP. Antibody specificity was confirmed with the use of two clonal CHO-K1 cell lines, stably transfected with the pEGFP-C2 vector containing coding murine ADFP and Tip47 sequences. The presence of ADFP and Tip47 in fat cake or total cell extract was analyzed by immunoblotting by loading 20 µg of total protein per lane onto 4-12% BisTris gradient mini gels (NuPAGE, Invitrogen). Immunostaining revealed that the ADFP and Tip47 antibodies do not cross-react (supplemental Fig. 1). Bodipy 558/568 and Bodipy 493/503 (Invitrogen) were used to visualize intracellular neutral lipid droplets. Alexa Fluor 488 and Alexa Fluor 598 species-specific secondary antibodies were purchased from Molecular Probes (Invitrogen). For loading control, anti-
-actin antibody was used (Abcam).
Immunocytochemistry and Immunoblotting—For ADFP and Tip47 staining, cells were fixed in 3% (v/v) paraformaldhyde for 15 min at room temperature. After a brief rinse in PBS, pH 7.4, cells were blocked before staining with donkey chromopure IgG or goat chromopure IgG (Jackson ImmunoResearch Laboratories) in the presence of 0.1% w/v of saponin for 2 h at room temperature. Double-labeled immunostaining was performed overnight at 4 °C with the appropriate antibodies. Primary antibodies were used in different combinations and dilutions as follows: 1:500 dilution for rabbit anti-murine or goat anti-murine ADFP and 1:500 dilution for rabbit anti-murine Tip47. After four washes with PBS with 0.1% w/v saponin, cells were incubated for 1 h with appropriate secondary antibodies (Invitrogen): Alexa Fluor 488-conjugated donkey anti-rabbit (1:1000 dilution) and Alexa Fluor 594-conjugated donkey anti-goat (1:500 dilution). Cells were viewed with a confocal laser microscope (LSM510; Carl Zeiss MicroImaging, Inc.) using a 63x oil objective lens. Cellular extracts for immunoblotting were obtained by scraping cells in Laemmli sample buffer containing 5% SDS and 1 mM dithiothreitol. Each lane of an SDS-PAGE gel was loaded with protein derived from a single well of a 24-multiwell dish.
Cellular Lipolysis and Lipogenesis—Lipolysis was performed according to Tansey et al. (16) and Sztalryd et al. (19) where re-esterification of fatty acids in CHO-K1 cells was largely prevented by the inclusion of 5 µM triacsin C (Biomol, Plymouth Meeting, PA), an inhibitor of acyl-coenzyme A synthetase, in the medium. Given our earlier published methods, and published work from another laboratory using this level of triacsin C to block fatty acid esterification into triglycerides (TAGs) (16, 19, 38), 5 µM triacsin C was used without confirmatory measurements that re-esterification and TAG synthesis were suppressed. Triplicate wells were tested for each condition.
Lipid Extraction and Thin Layer Chromatography—The cell monolayer was washed with ice-cold PBS and scraped into 1 ml of PBS. The lipids were extracted by the method of Bligh and Dyer (28) in the presence of an internal neutral lipid standard mix containing 10 µg each of monoolein, diolein, triolein, free cholesterol, oleic acid, and cholesteryl oleate (Supelco) for band identification. Thin layer chromatography extractions were performed as described previously (29, 30). In some experiments, the phospholipid spot remaining at the origin in our solvent system was scraped and re-extracted twice with chloroform methanol (1/2) and dried down under nitrogen before being kept at -80 °C. Polar lipid species were separated on silica gel G (Analtech) plates using a one-dimensional double-development procedure from Igal et al. (38). Briefly, phospholipids were separated in a double one-directional solvent system. First, the plate was developed with chloroform-methanol-30% ammonium hydroxide-H2O (70:25:3.5:1.5, v/v) to 3 cm from the top. The residual solvents on the plate were evaporated, and the plate was rerun in the same direction in chloroform:methanol:glacial acetic acid:H2O (80:10:2:0.75, v/v). Pure lipid standards: phosphatidylcholine, phophoinositol, and phosphoethanolamine were run in parallel (Avanti lipids) and were added to the samples 10 µg each for identification purpose. Spots were scraped and radioactivity determined.
Statistical Analysis—Statistical significance was tested using a one-way analysis of variance analysis (GraphPad Sofware Inc.) or a two-tailed Student's t test.
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RESULTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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Up-regulation of Tip47 in ADFP Null MEFs—To explore the possibility of compensatory mechanisms regulating lipid drop-let formation in non-adipogenic cells and their functional consequences, we established MEF cell lines from 16-day embryos of ADFP null and wt mice, as described under "Experimental Procedures." Immunofluorescent staining and immunoblot analysis (Fig. 2) confirmed the absence of ADFP in established ADFP null cell lines. More importantly, Bodipy staining of neutral lipids clearly indicated that ADFP null cells could accumulate lipid droplets in response to fatty acid loading. With the use of our recently developed Tip47 antibody (see "Experimental Procedures" and supplemental Fig. 1), further analyses showed higher expression of Tip47 mRNA and protein in the ADFP null than wt cells (Fig. 3). Furthermore, immunofluorescence staining showed that Tip47 coated the lipid droplets of ADFP null cells (Fig. 4). In addition, we confirmed the higher expression of Tip47 protein in liver fat extracts from ADFP null mice compared with wt mice (Fig. 5).
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Down-regulation of Tip47 by siRNA reduced the number of lipid droplets formed after 12 h of incubation with 400 µM oleic acid (Fig. 7C). Although both ADFP and Tip47 were lacking or reduced, these cells continued to accumulate some lipid droplets as evidenced by Bodipy neutral lipid staining. Lipid droplets in ADFP null cells did not appear to differ in size from those in control cells, but most lipid droplets lacking both Tip47 and ADFP at their lipid droplet surface appear as a single droplet (supplemental Fig. 4). To confirm the observation of reduced lipid droplet accumulation in the absence of both ADFP and Tip47, we determined the metabolic fate of exogenous NEFA. ADFP null cells treated with Tip47 and control siRNA were exposed to 400 µM oleic acid, supplemented with a trace of 3H-labeled oleic acid. Total lipids were extracted after 1, 6, and 12 h, and TLC was used to separate NEFA, monoacylglycerides, diacylglycerides, triglycerides (TAG), and phospholipids (29, 30). No statistical difference was observed in NEFA uptake into total lipids (Fig. 8), but the distribution of [3H] oleic acid among the phospholipid and TAG fractions was altered by siRNA inhibition of Tip47 (Fig. 8 and supplemental Table 1). By 12 h, cells lacking both ADFP and Tip47 accumulated nearly 40% less TAG, leading to the conclusion that in the absence of both ADFP and Tip47, metabolism of NEFA storage shifted from the TAG to the phospholipid fraction. This suggests that Tip47 helps direct influx of NEFA to incorporation in TAG. In addition, we examined whether lack of Tip47 could perturb the composition of phospholipids species but found that both ADFP null cells treated with control siRNA or Tip47 siRNA preferentially incorporated oleic acid in the phosphatidylcholine fraction (supplemental Table 2). Tip47 siRNA increased the lipolysis of newly synthesized TAG (determined in the presence of triacsin C) only at early time points, 30 min and 1 h (Fig. 9).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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ADFP is always found at the lipid droplet surface and serves as a convenient marker for non-adipogenic fat depositions (31). However, its role in non-adipogenic tissues has been difficult to characterize.
Numerous hypothetical roles have been proposed for ADFP in non-adipogenic cells. First, it was suggested that ADFP increases cellular NEFA uptake. ADFP overexpression in COS-7 cells leads to increased uptake of NEFA and the atypical presence of ADFP at the plasma membrane (32). By contrast, subsequent experiments in all other types of cells have found ADFP exclusively associated with the lipid droplet (13). Evidence has been reported that ADFP can bind long chain NEFA and it has been postulated that the protein recruits NEFA to the lipid droplets to be rapidly processed into TAG (32). Furthermore, like perilipin, ADFP was suggested to protect against lipolysis but in non-adipogenic tissue (33). In mammary epithelial cells, it may participate in the formation and secretion of milk lipid droplets (17) or in transporting TAG to supply NEFA to type II epithelial cells for the production of lung surfactant (34). Fragmentary data implicate ADFP with the formation of ectopic fat depositions that occur with insulin resistance in liver and muscle. Indeed, ADFP was found to be up-regulated in steatotic livers from dyslipotrophic AZIP mice (35) and from obese mice (36), and it has been suggested that ADFP plays a role in the pathology of non-alcohol mediated fatty liver disease (36). More recently, in mammalian cells and insect cells it was proposed that ADFP and other PAT proteins may play a role in moving lipid droplets within the cytosolic compartment via binding to motor proteins (37). Unfortunately, in our fibroblastic cellular system, ADFP loss of function did not result in a defined phenotype.
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It has been previously reported that Tip47 behaves differently than ADFP in that Tip47 is able to exist both in the cytosol as well as at the surface of the lipid droplet (10, 14, 24). ADFP mainly targets the lipid droplet surface, and unbound ADFP protein is rapidly degraded (20). These differences in lipid droplet binding characteristics and/or stability in aqueous environment may be responsible for the lack of Tip47 overexpression observed in the ADFP null mouse tissues studied (26). Indeed, Tip47 Western blots in the earlier study were performed using total cellular homogenate and not protein extracts from isolated fat cake. This interpretation is further supported by the presence of increased amount of Tip47 in ADFP null liver fat cake. Two manuscripts are now reporting a fifth member of the PAT family protein (10, 39), which is specifically expressed in oxidizing tissues. This fifth PAT protein is highly expressed in heart and red muscle and induced in fasting liver.5 We have checked by Western blotting that this protein was not expressed in the ADFP null fibroblasts (results not shown), but we predict that it will be overexpressed in heart, muscle, and in fasted liver of the ADFP null mice, as a compensatory mechanism. By analyzing what has been so far published in the field of PAT proteins, it is becoming increasingly clear that the PAT protein family function to enhance the lipid drop-let formation by protecting it against lipolysis. The variety of existing PAT proteins, five to date, may therefore reflect the lipid metabolism utilization of a particular cell type. Fibroblastic cells are not particularly adapted to oxidize NEFA or to store them, but in cell culture, they use NEFA to build essentially membranes and among the PAT proteins only Tip47 and ADFP are necessary to coat the lipid droplets. Muscle, liver, and heart utilize NEFA for oxidation and may require at various times a different protein coating at the surface of the lipid droplets, and finally adipose tissue, which is the major organ for lipid storage, expresses specifically perilipin, tightly regulating the flux of NEFA. In conclusion, the NEFA utilization and storage requirements in specific cell types may dictate the type of the PAT protein coating on the lipid droplet.
Our data further support the role of Tip47 in lipid droplet metabolism and lead us to hypothesize that in absence of ADFP, Tip47 may compensate functionally for ADFP and that these proteins provide redundancy in metabolic pathways. The presence of Tip47 or ADFP at the surface of the lipid droplet did not appear to affect the apparent size or number of lipid droplets in this fibroblastic cell culture system which suggests that ADFP and Tip47 have similar abilities to package lipids.
It could also be hypothesized that corresponding to what has been described in perilipin null mice adipocytes (i.e. in the absence of perilipin, ADFP re-appears on the droplets) in non-adipogenic cells in the absence of ADFP, Tip47 appears on the droplets. These observations may reflect a hierarchy in the lipid droplet affinities of the various PAT proteins, from perilipin, the most tightly bound to the droplet, to ADFP and finally Tip47.
It was suggested that in adipogenic 3T3-L1 cells, lipid droplet coating will vary as a function of time and subcellular location (24); we demonstrate that in the absence of ADFP, Tip47 will remain at the lipid droplet surface in non adipogenic cells, independently of time and subcellular location.
Our data support strongly the hypothesis that Tip47, in the absence of ADFP, plays an important role in packaging lipids. However, it is important to note that our experimental procedures prevented us from following the kinetics of lipid droplet formation and that our present data might be hampered by the stability of the PAT protein when bound to the surface of the lipid droplet. Indeed to obtain maximum inhibition of Tip47, cells have to be studied after 5 days of plating, and such an experimental procedure prevents us from following the fate of the lipid droplets into a specific cell during that time. Hence, we do not know if the observed lipid droplets observed at day 5 in cells lacking both ADFP and Tip47 are actually droplets that were formed at a time when Tip47 was still binding to the drop-let, and then by the day 5 time point, Tip47 disappeared from the lipid droplet, leaving intact the already formed droplet. It is also possible that nascent lipid droplets fused together during these 5 days, and the resultant observed droplets are just trying to offer minimum surface area to maintain themselves in the cytosol.
In conclusion, the data herein reveal that fibroblastic cells derived from ADFP null mice are still able to form lipid droplets and that their ability for FA uptake and lipolysis remains unchanged as Tip47 becomes the most abundant protein at the surface of the lipid droplet. Lack of both ADFP and Tip47, however, resulted in altered lipid metabolism. Cells retained the ability to form lipid droplets but to a much lesser extent and shunted the utilization of exogenously added NEFA from triglycerides to phospholipids, the mechanism for such a switch remaining unknown. These data support the hypothesis that Tip47 plays an important role in lipid metabolism.
Our results validate the use of siRNA to inhibit expression of PAT family proteins and induce a loss of function phenotype in a non-adipogenic cell type. Moreover, they have begun to give information about the role of Tip47 in lipid droplet formation in a fibroblastic cell. Our data suggest that in pathophysiological states such as obesity and diabetes when the supply of fatty acids is high, the relative abundance of Tip47, ADFP, and now the most recently described PAT-1/MLDP protein in peripheral tissues may play a critical role in regulating the formation and turnover, and hence metabolic consequences, of ectopic fat. It will therefore be important to investigate cell specific mechanisms regulating the cell-specific expression and function of PAT proteins.
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FOOTNOTES |
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1-4 and Tables 1 and 2.
2 Present addresses: Military Casualty Research, Walter Reed Army Institute of Research, Silver Spring, MD 20910 and the Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814.
1 To whom correspondence should be addressed: GRECC/geriatrics, Veterans Affairs Medical Center, 10 North Greene St., Baltimore, MD 21201. Tel.: 410-605-7000 5417; Fax: 410-605-7913; E-mail: csztalry{at}grecc.umaryland.edu.
3 The abbreviations used are: ADFP, adipose differentiation-related protein; TAG, triacylglycerol; NEFA, non-esterified free fatty acids; Tip47, Tip47 cargo protein; siRNA, small interference RNA; CHO, Chinese hamster ovary; PBS, phosphate-buffered saline; MEF, mouse embryonic fibroblast; wt, wild type; GFP, green fluorescent protein; Bis-Tris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol.
4 G. Xu and C. Londos, unpublished data.
5 K. Dalen, T. Dahl, E. Holter, B. Arntsen, C. Londos, C. Sztalryd and H. I. Nebb, submitted for publication.
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ACKNOWLEDGMENTS |
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TOPABSTRACTINTRODUCTIONEXPERIMENTAL PROCEDURESRESULTSDISCUSSIONREFERENCES |
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