Characterization of the Lipid Droplet Proteome of a Clonal Insulin

Article pubs.acs.org/jpr

Characterization of the Lipid Droplet Proteome of a Clonal Insulinproducing β-Cell Line (INS-1 832/13) Sara Larsson,*,† Svante Resjö,†,‡ Maria F. Gomez,§ Peter James,∥ and Cecilia Holm† †

Department of Experimental Medical Science, Division of Diabetes, Metabolism and Endocrinology, Lund Univeristy, BMC C11, SE-221 84 Lund, Sweden ‡ Department of Plant Protection Biology, Swedish University of Agricultural Sciences, PO Box 102, SE-23053 Alnarp, Sweden § Department of Clinical Sciences, Malmö, Lund University, SE-221 84 Lund, Sweden ∥ Department of Immunotechnology, Lund University, SE-221 84 Lund, Sweden ABSTRACT: Lipids are known to play a crucial role both in the normal control of insulin release and in the deterioration of β-cell function, as observed in type 2 diabetes. Despite this established dual role of lipids, little is known about lipid storage and handling in β-cells. Here, we isolated lipid droplets from oleate-incubated INS-1 832/13 cells and characterized the lipid droplet proteome. In a total of four rounds of droplet isolation and proteomic analysis by HPLC−MS/MS, we identified 96 proteins that were specific to droplets. The proteins fall into six categories based on function or previously observed localization: metabolism, endoplasmic reticulum/ribosomes, mitochondria, vesicle formation and transport, signaling, and miscellaneous. The protein profile reinforces the emerging picture of the lipid droplet as an active and dynamic organelle involved in lipid homeostasis and intracellular trafficking. Proteins belonging to the category mitochondria were highly represented, suggesting that the βcell mitochondria and lipid droplets form a metabolic unit of potential relevance for insulin secretion. KEYWORDS: lipid droplets, β-cells, insulin, proteomics

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INTRODUCTION The strong association between obesity and type 2 diabetes has emphasized the role of lipids in the development of type 2 diabetes.1 Ectopic lipid deposition plays a major role in the development of both insulin resistance and islet dysfunction, the two hallmarks of type 2 diabetes.2 The mechanisms whereby lipids cause dysfunction in insulin target tissues and in insulin-producing β-cells remain incompletely understood. In contrast to the harmful effects of lipidsreferred to as lipotoxicitylipids are also required for normal function of the insulin-producing β-cells. Islets depleted of their triglyceride stores exhibit a severe defect in glucose-stimulated insulin secretion.3 Addition of exogenous fatty acids corrects this defect, indicating that a lipid-derived signal with a critical role in insulin secretion is normally generated from the lipid stores of β-cells.4 The identity of this signal as well as its targets remains elusive. Understanding the opposing role of lipids for β-cell function will require a better understanding of lipid handling in β-cells. However, knowledge in this area is still scarce and the majority of published studies have focused on lipases. Hormone-sensitive lipase (HSL) and adipose triglyceride lipase (ATGL) have been shown to be expressed in β-cells, and both have been implicated in the generation of lipid signals from triacylglycerols.5−7 The storage of triacylglycerols is confined to lipid droplets. These are ubiquitous organelles consisting of a hydrophobic core of neutral lipids such as triacylglycerol and © 2012 American Chemical Society

cholesterol esters delimited by a monolayer of phospholipids that is coated by various proteins. The most well-known of these proteins are the members of the PAT family, that is, perilipin, ADRP, S3−12 and TIP47.8,9 Although initially believed to be structural components, it is now well established that several of the lipid droplet-associated proteins are important regulators of lipid mobilization and lipid storage.9,10 Lipid droplets have been observed in β-cells.11,12 Two members of the PAT family, perilipin and ADRP, have been shown to be expressed in β-cells and to regulate lipolysis in these cells.13,14 Apart from perilipin and ADRP, however, the protein composition of lipid droplets in β-cells is essentially unknown. The aim of this study was to characterize the lipid droplet proteome of β-cells. To this end, we isolated and purified lipid droplets (LDs) from clonal β-cells cultured in the presence of oleic acid and identified the lipid droplet-associated proteins using HPLC−MS/MS.

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MATERIALS AND METHODS

Materials

Oleic acid and Percoll were purchased from Sigma. Fatty acidfree BSA was from Roche Diagnostics GmbH (Mannheim, Germany) and SDS-PAGE gels were obtained from Invitrogen Received: September 23, 2011 Published: January 24, 2012 1264

dx.doi.org/10.1021/pr200957p | J. Proteome Res. 2012, 11, 1264−1273

Journal of Proteome Research

Article

20000g at 4 °C where the underlying liquid was carefully removed, the LDs were stored at −80 °C until further analysis. The cytosolic fraction was also collected from the middle of the tube and used as a control.

(Carlsbad, CA). ECL reagents super signal were from Pierce and the nitrocellulose membrane, Hybond-C extra was from GE Healthcare, (Cambridge, U.K.). Alexa Fluor594 antibody was from Invitrogen and BODIPY 493/503 was from Molecular Probes (Invitrogen). SNAP-25 and calnexin antibodies were obtained from Abcam and the NaKATPase antibody was from Novus Biologicals, (Littleton, CO). The antibody against Golgi-specific Brefeldin A (BFA)-resistance Factor 1 (GBF1) was purchased from BD Transduction Laboratories and the COX IV antibody was from Cell Signaling Technology (Beverly, MA). Antibodies against ADRP and perilipin were both kind gifts from Constantine Londos National Institutes of Health, Bethesda, MD and the HSL antibody was an affinity purified rabbit anti rat HSL antibody made in-house.

Subcellular Fractionation

The supernatant not used for LD isolation was mixed with Percoll to a final concentration of 15% v/v Percoll. A gradient was generated by centrifugation at 48000g for 25 min at 4 °C. The plasma membrane (PM) fraction (top) and vesicle fraction (bottom) were collected and washed three times in homogenization buffer (0.25 mM sucrose, 1 mM EDTA, 5 mM hepes, 1 mM dithiothreitol, pH 7.4 and 20 μg/mL leupeptin, 10 μg/mL antipain and 1 μg/mL pepstatin) by centrifugation at 150000g at 4 °C for 30 min. The pellets were resuspended in small volumes of homogenization buffer and 10 μg from each fraction was analyzed on Western blot for different subcellular fractionation markers. To obtain positive markers for Golgi, ER and mitochondria, the homogenate from 5 × 10 cm plates were fractionated as described by Göransson et al.20

Cell Culture and Incubation Conditions

INS-1 832/13 cells15 were cultured in RPMI 1640 containing 11.1 mM D-glucose supplemented with 10% heat inactivated fetal bovine serum, 100 IU/mL penicillin, 100 μg/mL streptomycin, 10 mM hepes, 1× glutamax, 1 mM sodium pyruvate and 50 μM β-mercaptoethanol, at 37 °C in a humidified atmosphere containing 5% CO2 and 95% air. Fortyeight hours before harvest, the medium was changed to either RPMI 1640 containing 1% fatty acid-free BSA or 0.5 mM oleate bound to 1% fatty acid free BSA, prepared as previously described,16 yielding a total and free concentration of oleate of 0.5 mM and 30 nM, respectively.17

Western Blot

LD proteins were acetone precipitated before Western blot analysis and were then dissolved in homogenization buffer before the entire fraction was subjected to a 4−12% polyacrylamide gel. Proteins were electroblotted to a nitrocellulose membrane and Western blot analysis was performed using the ECL system. Fractionation markers used were NaKATPase (PM), HSL, ADRP and perilipin (LD), calnexin (ER), GBF1 (Golgi) and COX IV (mitochondria).

Fixation and Staining of 832/13

Cells were grown on sterilized glass coverslips and incubated as described above. Cells were rinsed in PBS and fixated in 4% formaldehyde for 15 min, followed by blocking in 1% donkey serum, 1% BSA and 0.1% triton-X for 30 min. For identification of individual cells, preparations were stained for SNAP-25 (1:2000 in blocking buffer overnight at 4 °C), a plasma membrane bound protein. After rinsing in PBS, secondary antibody conjugated to Alexa Fluor594 was added for 1 h at room temperature. BODIPY 493/503, a lipophilic dye, was diluted in PBS to a final concentration of 3.8 μM and was added to the cells for 15 min followed by serial washings in PBS. BODIPY and SNAP-25 staining were visualized using a Zeiss LSM 5 Pascal laser scanning confocal microscope. In all images, the number of LDs per cell was counted to allow comparison of lipid content in control cells vs oleate-treated cells.

Insulin Measurement of Subcellular Fractions

An aliquot from each subcellular fraction was taken for measurement of insulin using ELISA (Novo Nordisk, Copenhagen, Denmark). Tryptic Digestion

Protein samples were boiled with SDS loading buffer and separated by 1D SDS-PAGE using 10% polyacrylamide. The electrophoresis was stopped, when the front had migrated 5 cm into the separation gel. The gels were stained with Coomassie Brilliant Blue and the entire protein lane was cut out from the gel and cut into smaller pieces. The gel pieces were washed with 100 mM ammonium bicarbonate followed by acetonitrile (ACN) and dried by rotary evaporation in a Speedvac. Cysteine residues were reduced with DTT and alkylated with iodoacetamide. The gel pieces were then subjected to two further rounds of washing and drying, followed by trypsin digestion overnight. The tryptic peptides were extracted and concentrated to a volume of approximately 50 μL using rotary evaporation.

Isolation of LDs

LDs were isolated using a published protocol,18,19 with some minor modifications. Briefly, cells of 18 × 150 mm plates were harvested in PBS containing 20 μg/mL leupeptin, 10 μg/mL antipain and 1 μg/mL pepstatin and centrifuged at 500g for 5 min at 4 °C. The pellet was resuspended in 20 mL of buffer A (25 mM tricine, 250 mM sucrose, pH 7.6 and 20 μg/mL leupeptin, 10 μg/mL antipain and 1 μg/mL pepstatin) and homogenized by N2 cavitation, 350 psi for 15 min on ice. The homogenate was centrifuged at 1000g for 10 min, and 14 mL of the supernatant was transferred to two SW41 tubes and the rest was saved for further fractionation. On top of each tube 3.5 mL buffer B (100 mM KCl, 2 mM MgCl2, 20 mM hepes pH 7.4) was added before centrifugation at 274000g for 1 h at 4 °C. The LD fraction appeared as a diffuse white band on top of the gradient and was carefully collected into a 0.5 mL tube. After concentration by four to five serial centrifugation steps at

Mass Spectrometry and Data Analysis

Before analysis by mass spectrometry, samples were desalted using a C18 StageTip, 200 μL tip model (Proxeon Biosystems) modified by the addition of Poros Oligo R3 material (Applied Biosystems) on top of the StageTip C18 material. The volume of Poros R3 was approximately equal to the volume of the stage tip C18 material. The tip was then washed with 80% ACN, 0.5% acetic acid (elution buffer), equilibrated with 3% ACN, 0.5% acetic acid, 1% trifluoroacetic acid (washing buffer) before the sample was applied, washed with washing buffer and eluted with elution buffer. The eluate was subjected to rotary evaporation in a speedvac to remove ACN and reduce the 1265



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volume. After speedvacing the volume was adjusted to 8 μL. Six microliters of the sample was subjected to HPLC−MS/MS analysis using an Eksigent nanoLC2D HPLC system coupled to an Orbitrap XL. The peptides were loaded onto a precolumn (Agilent Zorbax 300SB-C18, 0.3 mm ID, 5 mm, 5 μm particle size) connected to an analytical column (Agilent Zorbax 300SB C18, 75 μm ID, 150 mm, 3.5 μm particle size). The analytical column was pre-equilibrated for 10 min using buffer consisting of 0.1% formic acid (FA), 5% ACN at a flow rate of 10 μL/min and the peptides were separated in an 0.1 FA buffer using a 55 min linear gradient from 5 to 40% ACN followed by a 5 min linear gradient from 40 to 80% ACN, at a flow rate of 350 nL/ min. The eluted peptides were analyzed using an LTQ Orbitrap. The Orbitrap was operated in data dependent mode to automatically perform Orbitrap-MS and LTQ-MS/ MS analysis. Survey scan spectra (400−2000 Da) were acquired using the Orbitrap mass analyzer with the resolution R = 60000. Automatic gain control was enabled. The seven most intense ions were selected for fragmentation in the LTQ, using a mass window of 2 Da for precursor ion selection. The precursor ions were fragmented with a normalized collision energy of 35 (with activation Q set to 0.25 and an activation time of 30 ms). Dynamic exclusion was enabled with a repeat count of 2, a repeat duration of 20 s, an exclusion duration of 120 s, an exclusion list size of 499 and a 10 ppm exclusion mass width relative to both low and high. The raw data from the Orbitrap was converted to mgf files using ProteoWizard.21 The Proteios software environment22 was used to search the files with Mascot against the IPI rat database (version 3.71) extended with an equal size random part, with conserved protein length and amino acid distribution for the random part. Search tolerances were set to 12 ppm for MS and 0.4 Da for MS/MS. One missed cleavage was allowed. Carbamidomethylation of cysteine residues was selected as a fixed modification and oxidation of methionine residues was selected as a variable modification. Proteios was used to identify hits at an estimated false discovery rate of 0.01 using the random database mentioned above. Peptide identifications with a Mascot score >18 were considered significant. The MS data and protein identifications were exported to the PRIDE XML format and uploaded to http://www.ebi.ac.uk/pride/.23 Further information regarding this data set can be found there. The results of each HPLC−MS analysis can be viewed by searching for the accession number of the analysis of interest, as follows: 19827, cytosol control run 1; 19828, cytosol control run 2; 19829, cytosol control run 3; 19830, cytosol control run 4; 19831, LD run 1; 19832, LD run 2; 19833, LD run 3; 19834, LD run 4. Protein identifications from the LD samples were compared to the identifications from the control samples using the Hits Comparison feature of Proteios to identify proteins present only in the LD samples. In addition, all peptides assigned to proteins found to be unique for the LD fractions were checked manually against the search results from the cytosol fraction runs. This was done since a single identified peptide can occasionally be assigned to different proteins in different searches, if it matches more than one protein in the database (as is often the case for families of related proteins). Any proteins containing peptides found over the level of significance in the cytosol runs were then removed from the list of unique LD proteins. Additionally, a number of keratins identified were removed from the list since those identifications are likely to be the result of sample contamination.

Article

RESULTS

Isolation and Characterization of LDs

The number of LDs in β-cells is under normal conditions very low and in order to increase this number, cells were incubated for 48 h with 0.5 mM oleate. Staining with BODIPY 493/503 showed a 3.4-fold increase in number of LDs after oleate treatment compared to control cells incubated with 1% BSA (Figure 1A, B and C). In order to isolate LDs, a protocol

Figure 1. Representative confocal images showing INS-1 832/13 cells after 48 h incubation with either (A) 1% BSA or (B) 0.5 mM oleate. For identification of individual cells, samples were stained with an antibody against SNAP-25 (red), which yields a predominantly membrane-bound staining (white arrow in A). LDs are shown in green, as exemplified by the white arrowheads in B. Scale bars = 10 μm. (C) Summarized data from confocal experiments showing increased number of LD after treatment of cells with oleate as in B. In each image, the number of LDs was determined and normalized to the number of cells. Results are means of 20−22 images per treatment, each image including approximately 120−200 cells. Experiments were performed twice.

established for CHO cells was used. Besides this protocol, a protocol generating a vesicle and PM fraction was used (Figure 2). To confirm that the protocols worked satisfactorily the different fractions were analyzed using Western blot with markers for LDs and PM. As protein markers for LDs HSL, ADRP and perilipin were chosen. All three were found in the LD fraction from cells incubated for 48 h with 0.5 mM oleate but not in the LD fraction from control cells (Figure 2). HSL and perilipin were also found in plasma membrane fractions. NaKATPase was used as plasma membrane marker and was only found here and in the total cell homogenate, confirming the purity of this fraction. Insulin was used as a marker for the vesicle fraction, and was measured using ELISA. There was no detectable insulin in the cytosol and LD fractions, respectively, demonstrating that there was no vesicle contamination in these fractions. The insulin concentration of the plasma membrane fraction and the total homogenate was 50 ng/mL each, to be compared to 850 ng/mL in the vesicle fraction. The low concentration of insulin in the plasma membrane fraction presumably reflects a small fraction of insulin granules docked to the plasma membrane via an interaction of the SNARE proteins VAMP-2 on the vesicle and SNAP-25 and syntaxin 1 on the plasma membrane and that this interaction remains 1266



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Figure 3. A Venn diagram illustrating similarities of proteins found in four different LD isolations.

Characterization of the LD Proteome

A total of 96 proteins were identified uniquely in the LD fractions, i.e. they were identified in at least one of the four LD preparations and were absent from the control cytosolic fraction. These proteins were classified into 6 groups based on function or previously observed localization; metabolism, endoplasmic reticulum/ribosomes, mitochondria, vesicle formation and transport, signaling and miscellaneous. The 96 proteins identified in these 6 categories are listed in Table 1. In agreement with previous analyses of LD proteomes, proteins with defined roles in lipid metabolism were among the most common. In this category we found 22 proteins involved in fatty acid transport, triglyceride metabolism and maintenance of the LD, respectively. With regard to the latter group, we identified ADRP in all four replicates, whereas perilipin was not observed. Perilipin was, however, detected upon Western blot analysis of the LD fraction (Figure 2). With regard to lipases, ATGL was found in 4 of the replicates and its coactivator CGI58 was found in one of them, whereas HSL was not found, although detected by Western blot analysis (Figure 2). In the category endoplasmic reticulum/ribosomes we detected 26 proteins, reflecting the previously described link between the ER and the LD.24,25 Included among the ER proteins were chaperones, proteins involved in translation and reticulon proteins. In the category mitochondria we identified 16 proteins, which is a relatively larger number than that reported for most other LD proteomes (see below). This indicates a direct interaction between mitochondria and LDs, as has previously been described for steroidogenic cells,26,27 adipocytes,28 NIH 3T3 fibroblasts29 and myocytes.30 Many proteins involved in intracellular trafficking were identified on the β-cell LDs, including 6 members of the Ras related proteins, representing the endocytotic pathway as well as exocytotic pathway. In the trafficking category, also secretagogin, a EF-

Figure 2. (a) Schematic figure illustrating the 3 different subcellular fractionations made. (b) Western blot analysis of purified subcellular fractions. NaKATPase was used as a plasma membrane marker and HSL, perilipin and ADRP were all used as LD markers. Ten micrograms of protein were loaded on a gel for all fractions except for the LD fractions where all of the material was loaded. (c) Western blot run to illustrate that the LD fraction is pure from ER, Golgi and mitochondria proteins. Calnexin was used as ER marker, GBF1 as Golgi marker and COX IV as mitochondria marker. Ten micrograms of protein were loaded on a gel for all fractions except for the LD fraction where the whole fraction was loaded.

throughout the fractionation procedure. To further verify the purity of the LD fraction used for proteomics analyses it was analyzed against a mitochondrial marker (COX IV), an ER marker (calnexin) and a Golgi marker (GBF1) using Western blot. Positive controls for the different organelles were generated through a separate subcellular fractionation protocol (Figure 2). There was no detectable signal for markers of mitochondria, ER or Golgi in the LD fraction (Figure 2), thus confirming the purity of LD fraction. Reproducibility of Protein Identification

To assess the reproducibility, four different LD fractions were individually processed, including cell culturing, isolation, protein separation by one-dimensional gel electrophoresis, trypsinization and analysis by HPLC−MS/MS analysis. Since we only could detect the LD fraction markers HSL, ADRP and perilipin by Western blot in the cells incubated with oleate, we decided to exclude the control cells and continue with oleate treated cells alone in the HPLC−MS/MS analysis. Of the 96 proteins that were identified in these reproducibility experiments, 27 (28%) were common to all four replicates (Figure 3). 1267



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Table 1. LD Associated Proteins Found in 832/13 Cells Compared to the LD Proteome of Other Mammalian Cells protein

IPI number Wb = Western blot found in number of runs

references

Metabolism ADRP ATGL CGI 58 Probable saccharopine dehydrogenase (CGI 49) Perilipin Long-chain-fatty-acid-CoA ligase 1 Long-chain-fatty-acid-CoA ligase 3 Long-chain-fatty-acid-CoA ligase 4 Long-chain-fatty-acid-CoA ligase 6 Membrane-associated progesterone receptor component 1 NADH-cytocrome b5 reductase 3 Sterol-4-alpha-carboxylase-3-dehydrogenase, decarboxylating Lanosterol synthase 3-beta-hydroxysteroid dehydrogenase type 7 17-beta-hydroxysteroid dehydrogenase type 7 Lysophosphatidylcholine acyltransferase 1 Putative uncharacterized protein Lpcat4 Dehydrogenase/reductase (SDR family) 1 Hormone sensitive lipase Squalene epoxidase Trifunctional enzyme subunit alpha, mitochondrial Trifunctional enzyme subunit beta, mitochondrial Uncharacterized protein Hsd17b4 protein

IPI00365035 IPI00370366 IPI00417754 IPI00372804 Wb IPI00188989 IPI00205908 IPI00210503 IPI00324041 IPI00480820 IPI00231662 IPI00360954 IPI00566093 IPI00205271 IPI00327252 IPI00365394 IPI00372813 IPI00202971 Wb IPI00202028 IPI00212622 IPI00198467 IPI00197180 IPI00326948 Endoplasmatic reticulum/ribosomes Ubiquitin fusion degradation protein 1 homologue IPI00195248 Uncharacterized protein IPI00193949 PREDICTED: ribosome binding protein 1, isoform 3 IPI00763161 Transmembrane protein 109 IPI00372499 Transmembrane emp24 protein transport domain containing 9 IPI00364707 Transmembrane emp24 protein transport domain containing 10 IPI00231659 Toll-like receptor adaptor molecule 2 (Predicted) isoform CRA_a IPI00365118 Calnexin IPI00199636 Protein ERGIC-53 IPI00210116 UBX domain-containing protein 4 IPI00371952 Nuclear protein localization protein 4 homologue IPI00191492 Ribophorin 1 IPI00204365 Reticulon-4 IPI00230986 Sarcoplasmic/endoplasmic reticulum calcium ATPase 1 IPI00190020 Hypoxia up-regulated protein 1 IPI00210975 Uncharacterized protein IPI00208495 Ancient ubiquitous protein 1 IPI00196841 Choline-phosphate cytidylyltransferase B IPI00214821 NADPH-cytochrome P450 reductase IPI00231200 FAS-associated factor 2 IPI00948062 Dolichyl-diphosphooligosaccaride- protein glycosyltransferase subunit 2 IPI00188059 Similar to Ribosome-binding protein 1 IPI00188079 Polyadenylate-binding protein 1 IPI00189074 Similar to 60S ribosomal protein L12 IPI00210164 60S ribosomal protein L18 IPI00230917 Translocon-associated protein subunit alpha IPI00364884 Mitochondria ATPsynthase subunit alpha, mitochondrial IPI00396910 ATPsynthase subunit beta, mitochondrial IPI00551812 ATP synthase gamma chain IPI00454288 Cytochrome b-c1 complex subunit 2, mitochondrial IPI00188924 VDAC1 IPI00421874 VDAC2 IPI00198327 Solute carrier family 25 member 3, mitochondrial IPI00209115 Similar to phosphatidyl- glycerophosphate synthase, isoform CRA_a IPI00361551 1268

4 4 1 4

14,18,19,25,35,40−45,54,63 18,19,32,41,42,45 40−43 42−45 40,46

1 4 4 1 2 4 4 4 3 2 4 4 4

40,42 35,18,19,40−43,45 18,19,35,40,42,45,46

55 18,19,35,40−45 35,40−42,45 18,19,35,40−46 42 18,19,40,41,43 64

18,19,40,42,45,46 40,42,46

2 2 2 2 1

18,19,41,43

4 1 3 2 4 1 1 2 3 3 3 2 2 2 2 1 4 1 2 4 1 1 1 1 1 1

42

4 4 2 3 3 3 4 3

42

42 42

42 42

40,42,43,45,46,54,55 42 42,45

40,46 42,45,55 42 42,54

40,42−45,52,53 42 42,45 42,45 42,55

42 42

40,42,45

42 18,19,42,43 42



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Table 1. continued protein

IPI number Wb = Western blot found in number of runs

references

Mitochondria Prohibitin IPI00211756 Prohibitin-2 IPI00190557 ADP/ATP translocase 2 IPI00200466 Mitochondrial inner membrane protein IPI00364895 Leucine-rich PPR motif-containing protein, mitochondrial IPI00360075 Glutamate dehydrogenase 1, mitochondrial IPI00324633 NADH dehydrogenase [ubiquinone] flavoprotein 3, mitochondrial isoform 1 IPI00197986 Glyceraldehyde-3-phosphate dehydrogenase, mitochondrial IPI00199663 Vesicle formation and transport Ras-related protein Rap 1B IPI00187747 Ras-related protein Rab-2A IPI00202570 Ras-related protein Rab-5A IPI00210733 Ras-related protein Rab-7A IPI00215564 Ras-related protein Rab-11B IPI00210381 Ras-related protein Rab-18 IPI00198316 Enthoprotin IPI00454532 Putative uncharacterized protein Enth IPI00394532 Tumor protein p63-regulated gene 1-like protein IPI00877362 Vesicle-associated membrane protein-associated protein B IPI00209283 SAR1 gene homologue A (S. cerevisiae), isoform CRA_b IPI00372857 Signaling Guanine nucleotide-binding protein G(o) subunit alpha IPI00204843 Uncharacterized protein IPI00476573 Fchsd2 protein IPI00368464 Miscellaneous Alg2 protein IPI00367294 Ac22−233 IPI00369234 Methyltransferase like 7A IPI00654464 Histone H1.2 IPI00231650 Tmed4 protein IPI00212516 rRNA 2-O-methyltransferase fibrillarin IPI00208091 Dock8 protein IPI00187920 Receptor expression-enhancing protein 6 IPI00365960 Neuroendocrine convertase 2 IPI00204864 Uncharacterized protein IPI00392249 Chromodomain helicase DNA-binding protein 3 short isoform IPI00369880 Lman2 protein IPI00210524 Ubiquitin carrier protein IPI00371850 Selenocysteine insertion sequence-binding protein 2 IPI00212699 48 kDa protein IPI00209373 Lysosome membrane protein 2 IPI00231478 143 kDa protein IPI00366327 Cathepsin D IPI00212731

hand Ca2+-binding protein, was found. With regard to signaling proteins, only three were detected in our analyses of the LD proteome of β-cells.

1 2 4 1 2 1 1 1

40,42

1 4 4 1 4 2 1 3 4 2 1

35,42

18,19,42 42 42 42 42

18,19,42,55 42,54 18,19,40−46,54 18,19,42−44 18,19,40−44,57−59

42 42,55

2 1 3 4 4 4 3 2 2 1 1 1 2 1 1 1 2 1 1 1 4

42,45 42

55

55

42

proteins detected in the LD fraction were excluded because they were found in both the LD and control fractions. This exclusion of proteins found in both fractions is the reason for the absence of some abundant proteins detected in previous studies of the LD proteome (e.g., 14-3-3 protein, cofilin 1 and calreticulin). In total, 27 proteins were uniquely identified in the LD fraction of all four isolates. Most of these 27 proteins have previously been shown to reside on LDs, but some proteins were, to our knowledge, identified on LDs for the first time. In the category lipid metabolism we identified ADRP, but none of the other members of the PAT family. Using several different techniques, that is, Western blot analysis, qPCR and immunocytochemistry, we have previously identified perilipin in clonal as well as primary β-cells13 and also in the present

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DISCUSSION In this study we isolated LDs from oleate-incubated INS-1 832/ 13 cells using a modification of a protocol established for the isolation of LDs from CHO cells. The proteome of the isolated LDs was characterized and compared to the proteome of a cytosolic control fraction. Using this procedure, we identified 96 proteins unique to the LD fraction. All of the 96 proteins were not identified in all four isolates (see Figure 3 for details), but we believe that this variation is explained by the considerable technical difficulties involved in processing the small amount of LDs available. Furthermore, more than 90 1269



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hydroxysteroid dehydrogenase type 7 in three of the LD preparations is in agreement with reports from different cell types,18,19,40−43 including skeletal muscle, hepatocytes, adipocytes and CHO cells, and indicates that steroidogenesis occurs locally in β-cells in line with a few recent reports.47,48 In the category endoplasmic reticulum/ribosomes, at least five proteins involved in protein ubiquitination and proteasomal degradation were identified in three of four of the replicates; ubiquitin fusion degradation protein 1 homologue, FAS-associated factor 2, UBX domain-containing protein 4, nuclear protein localization protein 4 homologue and ancient ubiquitous protein 1. A connection between LDs and the ubiquitin/proteasome system has been suggested in several other studies. It has been proposed that LDs serve as storage depots for aggregation-prone proteins on their way to degradation.49,50 A model has been proposed suggesting that the lipid rearrangements required to form LDs facilitates movement of misfolded proteins from the ER to the cytoplasm.51 Ancient ubiquitous protein 1 has recently been identified on LDs of both A431 and HuH7 cells using immunohistochemical techniques and Western blot analysis of isolated droplets.52 An even more recent study demonstrates that ancient ubiquitous protein 1 in addition to its role in endoplasmic reticulum protein quality control also appears to be a determinant of LD formation, as its expression affected the level of LDs.52,53 In the category vesicle formation and transport, Rab-2A, Rab5A, Rab-11B, Tumor protein p63-regulated gene 1-like protein and putative uncharacterized protein Enth were the most reproducibly detected proteins, found in at least 3 of the replicates. Rab proteins have previously been identified on LDs18,19,40−46,54,55 and our results confirm that this is the case also in β-cells. Several studies show that Rab proteins are involved in membrane trafficking (for review, see ref 56) and the presence of Rab proteins on LDs suggests that LDs play a role in membrane trafficking. In fact, Rab 18, which was identified on LDs in two out of four runs in the present study, has in recent studies been shown to be involved in regulating the interaction of ER with LDs as well as the level of neutral lipid in the cell.57−59 Several studies have demonstrated a role for Rab proteins in the regulation of insulin secretion. For instance it has been shown that overexpression of Rab-2 in INS-1 cells delays transport of newly synthesized granule components, resulting in slow granule biogenesis and reduction in insulin secretion.60 Furthermore overexpression of the GDPbound or GTP-bound form of Rab-11 has been shown to inhibit insulin secretion in MIN6 cells, demonstrating the importance of the GTP/GDP cycle of Rab-11 in insulin secretion.61 Future studies will have to address the exact role of the different Rab proteins and other proteins involved in intracellular trafficking for β-cell function and dysfunction. The most striking feature of the LD proteome of the INS-1 832/13 cells described in this study was the high content of mitochondrial proteins, i.e17% of the proteins were found to belong to this category. A high representation of mitochondrial proteins on the LD was also found in a very recent study performed on cultured, oleate-incubated myoblasts, where 20% of the proteins were found to belong to this category.42 Using fluorescence microscopy and transmission electron microscopy a direct physical link between LD and mitochondria was also demonstrated in that study. An association between LD and mitochondria has previously been described also for adipocytes,28 NIH 3T3 fibroblasts,29 oocytes26and myo-

study we detected perilipin in the LD fraction using Western blot analysis. The reason why perilipin was not detected in the proteomic analyses is not known, but one possibility is that its abundance in β-cells under the conditions used is below the level of detection. The presence of ADRP in the LD fraction is in agreement with a recent report that used immunocytochemistry to show that ADRP is localized on the surface of LDs in murine pancreatic β-cells.14 The same study also showed that ADRP modulates the ability of fatty acids to acutely stimulate insulin secretion. The fact that the two other PAT proteins besides perilipin and ADRP, that is, TIP-47 and S3−12, were not detected in our study does not exclude that they may be expressed under certain metabolic conditions as shown in the adipocyte cell line 3T3-L1.31 ATGL was found on the LDs, whereas the other TG lipases shown to be expressed in islets, that is, HSL, adiponutrin and GS2,5,7 were not detected. ATGL and HSL have been shown to be the most highly expressed triglyceride lipases in islets7 and a location of ATGL at the LD is in agreement with findings from adipocytes.32 HSL, on the other hand, has been shown to be localized in close association with insulin granules in β-cells33 in contrast to the situation in adipocytes where HSL translocates from a cytosolic location to the LD in response to lipolytic stimulation.34 Although HSL was not discovered in the proteomic analysis of the LD fraction, it was detected by Western blot analysis. Since the LD fraction was shown to be devoid of contamination of insulin granules, this indicates that a small fraction of HSL indeed interacts with the LD surface under the experimental conditions of our study. Whether HSL translocates between different subcellular compartments in the β-cells, as has been shown for adipocytes, remains to be investigated. Two of the long-chain acyl-CoA ligases, also known as longchain acyl-CoA synthetases (ACS), were detected in all four replicates of LD fraction analysis, that is, ACS3 and ACS4, and two of them were found in one replicate, ACS1 and ACS6. ACS3 has previously been suggested to be a LD-related subtype of ACS, based on studies in HuH7 cells, where it was shown to redistribute from a perinuclear location to a location around the LDs during the course of LD formation.35 ACS1 and ACS4 are both subtype specific for mitochondria-associated membranes36 and their presence in the LD fraction of β-cells, suggests an interaction between mitochondria and LDs in these cells (see below). On the basis of the finding of both TG lipases and ACS enzymes on the LDs, it is tempting to suggest that acyl-CoA, proposed to be at the least one of the lipid-derived signals that act as coupling factor in the amplifying pathway of stimulussecretion coupling, is formed at the LD surface. As for fatty acids, a dual role for cholesterol in β-cells has emerged over the recent years. Cholesterol-enriched microdomains, which are likely to be formed through the action of HSL, exhibiting cholesterol ester hydrolase activity, have been shown to act as docking sites for insulin granules.37,38 Deposition of cholesterol esters, on the other hand, leads to β-cell dysfunction.39 In this context it is interesting to note that several enzymes involved in sterol metabolism were found in the LD fraction, in agreement with previous reports.18,19,35,40−46 The presence of lanosterol synthase, NADH-cytochrome b5 reductase 3 and the oxidoreductases, sterol-4-a-carboxylate 3-dehydrogenase and dehydrogenase/reductase 1, belonging to the SDR family, indicates that some steps of cholesterol biosynthesis occur at the LD. The finding of the steroidogenic enzymes 17-βhydroxysteroid dehydrogenase type 7 in two and 3-β1270



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cytes.30,62 The association has been demonstrated using different techniques; subcellular fractionation, confocal microscopy, electron microscopy and fluorescence resonance energy transfer (FRET). The fact that FRET can be used to demonstrate the association between mitochondria and LD indicates an association on a molecular scale with direct contact between the mitochondrial outer membrane and the monolayer of phospholipids surrounding the LD. In the present study the identification of proteins known to reside in the mitochondrial membrane, e.g. ATP synthase, Cytochrome b-c1 complex subunit 2, voltage-dependent anion channel 1 and 2 (VDAC 1 and 2), solute carrier family 25 member 3, ADP/ATP translocase and ACS4, in the LD fraction indicates a direct physical link between mitochondria and LDs in β-cells, although studies employing complementary methods are required to confirm this. In NIH 3T3 cells, the SNARE protein SNAP-23 was shown to mediate the interaction between mitochondria and LD and knockdown of SNAP-23 using siRNA reduced the formation of mitochondria-LD complexes as well as β-oxidation.29 The functional implications of a tight connection between mitochondria and LDs remain to be established but it can be speculated that this arrangement optimizes the delivery of fatty acids to mitochondria for βoxidation and for generation of acyl-CoA and other lipidderived coupling signals, whereas at the same time it minimizes the formation of harmful metabolic byproduct. In summary, our study reinforces the emerging picture of LDs as complex metabolically active organelles, harboring proteins and enzymes involved in storage and degradation of lipids, but also proteins involved in intracellular trafficking. Complete pathways do not appear to be present on LDs, which rather serve as repositories of raw materials and shuttles for lipid intermediates with bidirectional movement mediated by the rab proteins. The large representation of mitochondrial proteins in the LD fraction suggests a tight interaction between mitochondria and LDs forming a metabolic unit of importance for β-cell function.

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(3) Chen, G.; Koyama, K.; Yuan, X.; Lee, Y.; Zhou, Y. T.; O’Doherty, R.; Newgard, C. B.; Unger, R. H. Disappearance of body fat in normal rats induced by adenovirus-mediated leptin gene therapy. Proc. Natl. Acad. Sci. U.S.A. 1996, 93 (25), 14795−9. (4) Koyama, K.; Chen, G.; Wang, M. Y.; Lee, Y.; Shimabukuro, M.; Newgard, C. B.; Unger, R. H. beta-cell function in normal rats made chronically hyperleptinemic by adenovirus-leptin gene therapy. Diabetes 1997, 46 (8), 1276−80. (5) Mulder, H.; Holst, L. S.; Svensson, H.; Degerman, E.; Sundler, F.; Ahren, B.; Rorsman, P.; Holm, C. Hormone-sensitive lipase, the ratelimiting enzyme in triglyceride hydrolysis, is expressed and active in beta-cells. Diabetes 1999, 48 (1), 228−32. (6) Fex, M.; Olofsson, C. S.; Fransson, U.; Bacos, K.; Lindvall, H.; Sorhede-Winzell, M.; Rorsman, P.; Holm, C.; Mulder, H. Hormonesensitive lipase deficiency in mouse islets abolishes neutral cholesterol ester hydrolase activity but leaves lipolysis, acylglycerides, fat oxidation, and insulin secretion intact. Endocrinology 2004, 145 (8), 3746−53. (7) Peyot, M. L.; Guay, C.; Latour, M. G.; Lamontagne, J.; Lussier, R.; Pineda, M.; Ruderman, N. B.; Haemmerle, G.; Zechner, R.; Joly, E.; Madiraju, S. R.; Poitout, V.; Prentki, M. Adipose triglyceride lipase is implicated in fuel- and non-fuel-stimulated insulin secretion. J. Biol. Chem. 2009, 284 (25), 16848−59. (8) McManaman, J. L.; Zabaronick, W.; Schaack, J.; Orlicky, D. J. Lipid droplet targeting domains of adipophilin. J. Lipid Res. 2003, 44 (4), 668−73. (9) Brasaemle, D. L. Thematic review series: adipocyte biology. The perilipin family of structural lipid droplet proteins: stabilization of lipid droplets and control of lipolysis. J. Lipid Res. 2007, 48 (12), 2547−59. (10) Lass, A.; Zimmermann, R.; Oberer, M.; Zechner, R. Lipolysis - a highly regulated multi-enzyme complex mediates the catabolism of cellular fat stores. Prog. Lipid Res. 2011, 50 (1), 14−27. (11) Cnop, M.; Hannaert, J. C.; Hoorens, A.; Eizirik, D. L.; Pipeleers, D. G. Inverse relationship between cytotoxicity of free fatty acids in pancreatic islet cells and cellular triglyceride accumulation. Diabetes 2001, 50 (8), 1771−7. (12) Fex, M.; Nitert, M. D.; Wierup, N.; Sundler, F.; Ling, C.; Mulder, H. Enhanced mitochondrial metabolism may account for the adaptation to insulin resistance in islets from C57BL/6J mice fed a high-fat diet. Diabetologia 2007, 50 (1), 74−83. (13) Borg, J.; Klint, C.; Wierup, N.; Strom, K.; Larsson, S.; Sundler, F.; Lupi, R.; Marchetti, P.; Xu, G.; Kimmel, A.; Londos, C.; Holm, C. Perilipin is present in islets of Langerhans and protects against lipotoxicity when overexpressed in the beta-cell line INS-1. Endocrinology 2009, 150 (7), 3049−57. (14) Faleck, D. M.; Ali, K.; Roat, R.; Graham, M. J.; Crooke, R. M.; Battisti, R.; Garcia, E.; Ahima, R. S.; Imai, Y. Adipose differentiationrelated protein regulates lipids and insulin in pancreatic islets. Am. J. Physiol. Endocrinol. Metab. 2010, 299 (2), E249−57. (15) Hohmeier, H. E.; Mulder, H.; Chen, G.; Henkel-Rieger, R.; Prentki, M.; Newgard, C. B. Isolation of INS-1-derived cell lines with robust ATP-sensitive K+ channel-dependent and -independent glucose-stimulated insulin secretion. Diabetes 2000, 49 (3), 424−30. (16) Mulder, H.; Yang, S.; Winzell, M. S.; Holm, C.; Ahren, B. Inhibition of lipase activity and lipolysis in rat islets reduces insulin secretion. Diabetes 2004, 53 (1), 122−8. (17) Spector, A. A.; Fletcher, J. E.; Ashbrook, J. D. Analysis of longchain free fatty acid binding to bovine serum albumin by determination of stepwise equilibrium constants. Biochemistry 1971, 10 (17), 3229−32. (18) Bartz, R.; Zehmer, J. K.; Zhu, M.; Chen, Y.; Serrero, G.; Zhao, Y.; Liu, P. Dynamic activity of lipid droplets: protein phosphorylation and GTP-mediated protein translocation. J. Proteome Res. 2007, 6 (8), 3256−65. (19) Liu, P.; Ying, Y.; Zhao, Y.; Mundy, D. I.; Zhu, M.; Anderson, R. G. Chinese hamster ovary K2 cell lipid droplets appear to be metabolic organelles involved in membrane traffic. J. Biol. Chem. 2004, 279 (5), 3787−92. (20) Goransson, O.; Wijkander, J.; Manganiello, V.; Degerman, E. Insulin-induced translocation of protein kinase B to the plasma

AUTHOR INFORMATION

Corresponding Author

*Tel: + 46 46 2229772. Fax: + 46 46 2224022. E-mail: sara. [email protected].

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ACKNOWLEDGMENTS Financial support was provided by the Swedish Research Council (Grant 11284 to CH), the Swedish Diabetes Foundation, Novo Nordisk Foundation, A. Påhlsson Foundation, the European Union (Integrated Project EuroDia LSHMCT-2006-518153 in the Framework Programme 6[FP6] of the European-Community), the Swedish Heart and Lung Foundation (HLF20080843 to M.G.F), the Knut and Alice Wallenberg foundation and Lund University Diabetes Center. Support by BILS (Bioinformatics Infrastructure for Life Sciences) through Fredrik Levander is gratefully acknowledged.

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Characterization of the Lipid Droplet Proteome of a Clonal Insulin

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