Remodeling of Droplets during Lipolysis and Growth in Adipocytes

Margret Paar*1, Christian Jungst§1,2, Noemi A. Steinert, Christoph Magnes ~, Frank Sinner~, Dagmar Kolbll, Achim Lass*, Robert Zimmermann*, Andreas Zumbusch§, Sepp D. Kohlwein*, and Heimo Wolinski*3 From the *Institute of Molecular Biosciences, Lipidomics Research Center LRC Graz, University of Graz, 8070 Graz, Austria, the §Department of Chemistry, University of Konstanz, 78457 Konstanz, Germany, ~HEAL TH, Institute for Biomedicine and Health Sciences, Joanneum Research, 8036 Graz, Austria, and the IIlnstitute of Cell Biology, Histology and Embryology, and ZMF, Center for Medical Research, Medical University of Graz, 8070 Graz, Austria

Background: Micro-lipid droplets (mLDs) appear in adipocytes upon lipolytic stimulation. LDs may grow by spontaneous, homotypic fusion. Results: Scavenging of fatty acids prevents mLD formation. LDs grow by a slow transfer of between LDs. Conclusion: mLDs form due to overflow. I.D growth is a controlled process. Significance: Novel mechanistic insights into LD remodeling are provided.

Synthesis, storage, and turnover oftriacylglycerols (TAGs) in mation of large LOs requires a yet uncharacterized protein adipocytes are critical cellular processes to maintain lipid and machinery mediating LO interaction and lipid transfer. energy homeostasis in mammals. TAGs are stored in metaboli­ cally highly dynamic lipid droplets (LOs), which are believed to undergo fragmentation and fusion under lipolytic and lipogenic Most eukaryotic organisms deal with a typically fluctuating conditions, respectively. Time lapse fluorescence microscopy food supply by storing or mobilizing lipids as an energy source. showed that stimulation of lipolysis in 3T3-Ll adipocytes causes Malfunction of the synthesis or degradation of stores is progressive shrinkage and almost complete degradation of all linked to prevalent diseases, such as obesity, type II diabetes, or cellular LDs but without any detectable fragmentation into various forms of lipodystrophy (1). In mammals, adipose tissue micro-LOs (mLOs). However, mLOs were rapidly formed after functions as the major energy depot of the body. Excess fatty induction of lipolysis in the absence of BSA in the culture acids (FAs)4 and sterols are stored as neutral lipids (mostly as medium that acts as a fatty acid scavenger. Moreover, mLO for­ triacylglycerol (TAG) as well as steryl esters) in cytosolic lipid mation was blocked by the acyl-CoA synthetase inhibitor triac­ droplets (LDs), which are mobilized by regulated lipolytic sin C, implicating that mLOs are synthesized de novo in response breakdown in response to specific nutritional needs of the cell to cellular fatty acid overload. Using label-free coherent anti­ or organism. The LD surface consists of a phospholipid mono­ Stokes Raman scattering microscopy, we demonstrate that LOs layer harboring a set of enzymes and regulatory proteins that grow by transfer of lipids from one organelle to another. Nota­ catalyze the highly metabolically controlled synthesis and bly, this lipid transfer between closely associated LOs is not a mobilization of fat stores. Compartmentation of nonpolar neu­ ('apid and spontaneous process but rather occurs over several h trallipids into LDs ensures their physiological accessibility and, and does not appear to require physical interaction over large thus, plays a central and critical role in lipid homeostasis. Neu­ LO surface areas. These data indicate that LO growth is a highly trallipid turnover is accompanied by a significant remodeling regulated process leading to the heterogeneous LO size distri­ of LDs, and both the appearance of microlipid droplets (mLDs) bution within and between individual cells. Our findings suggest during breakdown of neutral lipids and growth of the organelles that lipolysis and lipogenesis occur in parallel in a cell to prevent by fusion events have been discussed as fundamental processes cellulai' fatty acid overflow. Furthermore, we propose that for- required for efficient mobilization and storage of fat in adi­ pocytes (2-6). A number of studies have reported that in differentiated * This work was supported in part by grants from the Austrian Science Funds, murine (3T3-Ll) adipocytes, large perinuclear LDs fragment FWF, Project LlPOTOX F300S-B12 (to S. D. K.) and Ph.D. Program Molecular Enzymology, Project W901 -BOS, and the Austrian Federal Government for and disperse into smaller mLDs in response to lipolytic stimu­ Science and Research (Project GOLD, in the framework of the Austrian lation (7- 10). Such a fragmentation process is expected to dras­ Genome Program GEN-AU) (to S. D. K. and H. W.). tically increase the surface/volume ratio of the LDs, leading to ,€ Author's Choice-Final version full access. more efficient degradation of stored neutral lipids by LD-asso­

1 Both authors contributed equally to this work. ciated lipases. Dispersion of LDs was also found to affect the 2 Supported by contract research "Methoden fUr die Lebenswissenschaften" locali zation of perilipin, one of the main regulators of LD turn- of the Baden-Wurttemberg Stiftung and a personal scholarship from the Konstanz Research School Chemical Biology. 3 To whom correspondence should be addressed: Institute of Molecular Bio­ 4 The abbreviations used are: FA, fatty acid; TAG, triacylglycerol; LD, lipid sciences, Lipidomics Research Center LRC Graz, University of Graz, Hum­ droplet; mLD, micro-LD; nLD, nano-LD; CARS, coherent anti-Stokes Raman boldtstrasse 50/11, 8010 Graz, Austria. Tel.: 43-316-380-5489; Fax: 43-316- scattering; ER, endoplasmic reticulum; ATGL, adipose lipase; 380-98S4; E-mail: heimo.wolinski@u ni-graz.at. NA, numerical aperture.

11164 over, as well as of hormone-sensitive lipase. Both proteins are associated LDs via a "bridge" between adjacent LDs and without restricted to a subpopulation of dispersed LDs, and it was sug ~ apparent spatial interaction over large LD surface areas. gested that mLDs that emerge under lipolytic conditions may represent an active pool of LDs from which neutral lipids are EXPERIMENTAL PROCEDURES mobilized (10). Controversially, however, a recent time lapse Cell Culture-Cells were cultured in glass bottom dishes with study using label-free coherent anti-Stokes Raman scattering a 50-mm diameter (MatTek Corp., Ashland, MA). 3T3-Ll (CARS) microscopy showed that in differentiated 3T3-Ll adi­ fibroblasts were grown in Dulbecco's modified Eagle's medium pocytes, mLDs appeared scattered throughout the cytosol upon (DMEM) containing 4.5 g/liter glucose and I.-glutamine (Invit­ lipolytic stimulation but were not detected at specific regions rogen) supplemented with 10% fetal calf serum (FCS) (Sigma­ neighboring large LDs as would be expected if they originated Aldrich) and antibiotics (DMEM + I +) under standard condi­ by LD fragmentation. It was suggested that these LDs may tions (37 DC, humidified atmosphere, 5% CO2) , Two days after instead derive from other organelles, such as the endoplasmic confluence, medium was changed to DMEM + I + containing reticulum (ER), rather than from existing LDs. On the other 10 J.Lg/ml insulin (Sigma-Aldrich), 0.25 J.LM dexamethasone hand, "nano-LDs" (nLDs), which may not be detectable by (Sigma-Aldrich), and 500 J.LM isobutylmethylxanthine (Sigma­ CARS microscopy, may split off from larger LDs and subse­ Aldrich). After 3 and 5 days, medium was changed to quently fuse to give rise to mLDs that are found dispersed in the DMEM +!+ containing 10 J.Lg/ml and 0.05 J.Lg/ml insulin, cytosol (11). respectively. The day before the experiment, cells were incu­ Furthermore, controversy also exists concerning the mode of bated without insulin overnight. Experiments were performed LD growth. Homotypic interaction between LDs of3T3-Ll adi­ on day 8 or 9 after initiation of differentiation. For electron pocytes may indeed cause fusion of the organelles; this process microscopy, cells were cultured on collagen-coated (1 % colla­ does not require TAG synthesis but depends on microtubules gen) Alcar film (Gropl, Inc., Tulln, Austria) placed in the glass and the motor protein dynein (12, 13). Furthermore, it was sug­ bottom dishes. gested that SNARE proteins mediate LD fusion (14). However, Human adipose-derived stem cells (Invitrogen) were grown in another study, fusion of LDs could not be observed in the in complete MesenPro RS Medium (Invitrogen), and after same cell type under conditions of induced lipid synthesis. reaching confluence, the medium was changed to Complete According to this study, nascent LDs may form at the cellular Adipogenic Differentiation Medium (Invitrogen). For long periphery and move toward larger perinuclear "storage" LDs. term experiments, cells were seeded in glass bottom dishes with Such LDs enlarge during their movement without observable a 35 -mm diameter (Ibidi, Germany) with an additional culture fusion events, presumably by synthesis of new TAG directly on insert (Ibidi) to enable four-dimensional CARS imaging over the LD (15). Both diacylglycerol and diacylglycerol acyltrans­ more than a week without the need for changing the medium. ferase 2 catalyzing the conversion of diacylglycerol to TAG Lipolytic Stimulation ofMurin e Adipocytes and Inhibition of were detected in the vicinity of lipid droplets both in 3T3-Ll Long Chain Fatty Acyl-CoA Synthetase- For stimulation of and COS7 cells, suggesting that LDs may also grow by biosyn­ lipolysis, 10 J.LM forskolin (Sigma-Aldrich) was added to the thesis of TAG near LDs and direct incorporation into existing medium. To study the effect of bovine serum albumin (BSA) on LDs (16). Moreover, it was shown that LDs of rat hepatoma cells LD forination during lipolysis, 3T3-Ll cells were incubated grow by incorporation of esterified into existing with 10 J.LM forskolin (in DMEM), either containing 2% fatty lipid droplets rather than by fusion events (17) . A recent anal­ acid-free BSA or no BSA. Incorporation of FAs into TAG was ysis of LD fusion in murine adipocytes using time lapse light inhibited by the addition of 5 J.LM triacsin C (Sigma-Aldrich). microscopy showed that fast organelle fusion can be stimulated First, cells were treated with triacsin C for 2 h in DMEM + 1+, by various drugs, which, however, appears to be a rare event in and then the medium was replaced by fresh DMEM containing untreated cells (18) . Finally, in addition to de novo synthesis and 5 J.LM triacsin C, 10 J.LM forskolin, and either 2% fatty acid free fusion, LDs may also grow by a dynamic interaction and gradual BSA (control) or no BSA. A total medium volume of 2 ml was (regulated) transfer of TAG between nascent and preformed used in all experiments. LDs, as shown in primary mouse hepatocytes. In these cells, Analysis ofAcyl -CoA Levels in Triacsin C-treated Differenti­ transient fusion and fission events may occur upon contact of ated 3T3-Ll Cells by Mass Spectrometry-Cells were differen­ two closely associated LDs (19). tiated for 8 days and subsequently pretreated for 2 h with vari­ In this study, we applied high resolution long term four-di­ ous concentrations of triacsin C (1, 5, 10, or 20 J.LM) to test the mensionallive cell imaging of murine adipocytes and human efficacy of the drug to inhibit fatty acid activation. After inhib­ adipose-derived stem cells to monitor the breakdown as well as itor treatment, cells were further incubated for 1 h in fresh the formation of LDs. Our results demonstrate that efficient DMEM again containing various concentrations of triacsin C degradation of LDs is not accompanied by fragmentation and and 2% BSA. Acyl-CoAs were determined by on-line solid dispersion of LDs in 3T3-Ll adipocytes but rather leads to FA phase extraction liquid chromatography-mass spectrometry as overflow that initiates formation of new LDs. This mLD forma­ described previously (20). In brief, buffer-suspended cells (250 tion can be prevented by excess BSA in cell culture medium to J.L1 of50%0.1 MKH 2P04 and 50% 2-propanol, prechilled to 4 DC) sequester lipolysis-derived FA or by inhibiting FA activation by were added to internal standard mix (0.25 nmol of 13C'6-palmi­ triacsin C, even in the abserice of extracellular FA acceptors. toyl-CoA, 0.25 nmol of 13C1s-stearoyl-CoA, 0.1 nmol of 13C'8- Long term monitoring of LD growth during adipocyte cultiva­ oleoyl-CoA per sample). After the addition of 15 J.Ll of saturated tion revealed a slow transfer of neutral lipids between closely aqueous (NH4)2S04 solution and 0.25 ml of acetonitrile, the cell

11165 suspension was homogenized on ice for 10 - 20 s using an ultra­ quency using the Stokes beam tuned to 998 nm with a power of sonic homogenizer. The homogenate was vigorously mixed and 6.5 milliwatts and the pump laser tuned to 777 nm with a power centrifuged at 2500 X g for 10 min at 4 "C, and the supernatant of62 milliwatts. A Leica 0.85 NA, X40 air objective was used for was transferred to autosampler vials. Extracts were stored at focusing the excitation beams. Data were recorded in transmis­ - 80 DC prior to analyses on an Ultimate 3000 System (Dionex, sion type geometry (forward CARS) and were collected by a LC Packings, Sunnyvale, CA) consisting of an autosampler with Leica 0.55 NA air condenser and transmitted through 641/75 cooled tray and a column oven with a switching unit coupled to and 680/SP (Semrock) emission filters. For long term experi­ an LTQ Orbitrap XL (Thermo Scientific, Waltham, MA). A ments, cells were incubated directly on the microscope stage, Phenomenex Strata X 2.0 X 20-mm cartridge (Torrance, CA) using a live cell-imaging chamber (Stage Top Incubator, Tokai and a Waters XBridge column (2.1 X 50 mm, 2.5 p,m) (Milford, Hit, Japan), and z-stacks of images were recorded with a voxel MA) were used for on-line solid phase extraction and as an size of260 X 260 X 400 nm (x/y/z). analytical column, respectively. Positive electrospray ioniza­ Transmission Electron Microscopy- Adipocytes were fixed tion-mass spectrometry was performed by high resolution mass in 0.1 M cacodylate buffer (pH 7.2) containing 2.5% glutardial­ spectrometry (scan range 500 - 1500 m/z, resolution 60,000). dehyde and 2% paraformaldehyde for 30 min at room temper­ Biochemical Analysis of Fatty Acid Release-For determina­ ature. Cells were rinsed twice in 0.1 M cacodylate buffer for 1 h tion of free FAs, aliquots of the corresponding media were col­ and postfixed in 2% osmium tetroxide in the same buffer for 1 h. lected, and free FA content was determined using a commercial After rinsing four times for 10 min in 0.1 M cacodylate buffer, analysis kit (W AKO Chemicals GmbH, Neuss, Germany). Cells specimens were dehydrated in a series of 50, 70, 90, and 100% were lysed in 0.3 M NaOH, 0.1 % SOS, and protein concentration cold acetone for 30 min each. Preparations were infiltrated by was determined using BCA reagent (Pierce). 2:1,1:1, and 1:2 mixtures of 100% acetone and agar 100 epoxy Fluorescence Microscopy- Imaging of fluorescently labeled resin (Gropl, Inc.) and pure agar 100 epoxy resin for 4 h. Finally, structures was performed on a Leica SP5 confocal microscope cells were placed in agar 100 epoxy resin at room temperature using a X40, NA 1.25 oil immersion objective. LOs of 3T3-Ll for 8 h, transferred into embedding molds, and polymerized at cells were labeled by adding L0540 (21) to the culture medium 60 DC for 4·8 h. Ultrathin sections (70 nm) were cut with a Leica (tlnal concentration 1 p,g/ml). The neutral lipid-specific dye Ultracut UC6 and stained with lead citrate for 5 min and with L0540 was a kind gift from Christoph Thiele (Max Planck Insti­ uranyl acetate for 15 min. Images were taken on a Tecnai 20 tute of Molecular Cell Biology and Genetics). Labeling of neu­ transmission electron microscope (Fei, Inc.). trallipids was typically achieved after 10 -15 min of incubation. Imaging-based Quantification of LD Size- A z-stack of L0540 fluorescence was excited at 561 nm, and emission was images of a selected cell was projected into a single two-dimen­ detected between 570 and 620 nm. For four-dimensional live sional image using maximum intensity projection. The diame­ cell imaging, cells were incubated in glass bottom dishes ter of - 50 LOs was measured using the line measure feature directly on the microscope stage using a PECON S-2 stage incu­ implemented in ImageJ (23), and surface and volume data were bator (PECON, Inc.) at 37 DC and 5% CO2 • Three-dimensional calculated. image data were acquired in 30-min time intervals and with a Statistical Analysis of FA Release- Data arc represented as voxel size of90 X 90 X 300 nm (x/y/z).100-nm TetraSpeck™ mean values and S.D. from three independent experiments. microspheres (Invitrogen) were used as subresolution particles Group differences were calculated using unpaired Student's t for determination of detection capabilities of the fluorescence test (two-tailed). Levels of statistical significance were consid­ imaging system (ll.ex/Aem' 561 nm/570 - 620 nm). ered as follows: p < 0.05 (' ), p < 0.01 (" ), and p < 0.001 (... ). CARS Microscopy- CARS microscopy of 3T3-Ll cells was performed using a commercial setup consisting of a picosecond RESULTS laser source and an optical parametric oscillator (OPO; pico­ Lipolytic Stimulation of 3T3-Ll Adipocytes Causes Rapid Emerald (APE (Berlin, Germany) and HighQ Laser (Rankwcil, Shrinkage of LDs without Detectable LD Fragmentation- To Austria)) integrated into a Leica SP5 confocal microscope investigate the effect of lipolysis on LO size and subcellular (Leica Microsystems, Inc.). Detection of the CARS signal was distribution, differentiated 3T3-Ll adipocytes were labeled achieved using 6501210 and 770/SP emission filters. The micro­ with L0540, stimulated with forskolin, and analyzed over time scope was equipped with a non-descanned detector for acqui­ by four-dimensional live cell imaging. Already within 30 min sition of signals in forward CARS mode (22) . To detect neutral after initiation of lipolysis, a clear shrinkage of LOs was detect­ lipids/LOs, the laser was tuned to 2845 cm - I , thus enabling able. Notably, all monitored LOs showed a progressive response imaging of CH2 symmetric stretching vibrations. For imaging, a to lipolytic stimulation, independent of their size or subcellular Leica 1.25 NA, X40 oil objective was used. For long term exper­ position (Fig. 1). Moreover, degradation of LO contents iments of 3T3-Ll cells, z-stacks of images were taken at differ­ appeared to be correlated with the size of the organelles. Com­ ent time intervals, as indicated, with a voxel size of 90 X 90 X puted values for the half-life of the volume of selected LOs dur­ 300 nm (x/y/z). ing lipolysis indicate that smaller LOs are faster degraded than Four-dimensional imaging of human adipose-derived stem larger LOs (Fig. 3A). Five hours after stimulation of lipolysis, cells was performed on a "home-built" CARS setup, based on a LOs in most cells were almost completely depleted. Quite nota­ multi photon microscope (Leica TCS SP5) and an Er:fiber laser bly, no fragmentation of existing LOs was detectable under our source (Toptica FemtoFiber Pro) at a repetition rate of40 MHz. experimental conditions, neither at the beginning of the exper­ The experiments were performed at 2845 cm - I resonance fre- iment nor at later stages, when the TAG stores were almost

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.~ I 2h ...... -., ·EJ FIGURE 1. Four-dimensional live cell imaging of LD consumption in 3T3-L 1 adipocytes. All imaged LOs decrease their size significantly within 2 h after stimulation of lipolysis by forskolin. In addition, the cell contract with progreSSing LO breakdown as indicated by the LOS40 "background" signal FIGUR E 2. Depletion of neutral lipid stores in 3T3-L 1 adipocytes. A, cell and decreased cell area. No fragmentation or dispersion of LOs is observed with almost totally depleted neutral lipid stores 5 h after stimulation of lipo­ during lipolysis. Left, maximum intensity projections of three-dimensional lysis using forskolin. An individual LO appears to be degraded faster than data acquired at the indicated time points; middle, enlarged area enclosed by other LOs of comparable size (solid arrows). LOs do not significantly alter their the white rectangular frame shown in the left panel; right, direct volume-ren­ subcellular position relative to each other during lipolysis (open arrows) . dering representations. Bar, 10 j.tm. B, 3T3-L 1 cells stimulated with isoproterenol and isobutylmethylxanthine instead of forskolin. Similar to forskolin stimulation, LOs shrink but without entirely depleted (Figs. 1 and 2A). Similarly, no mLDs were detectable fragmentation or dispersion. Maximum intensity projections are observed in differentiated 3T3-Ll adipocytes that were stimu­ shown of three-dimensional data acquired at the indicated time points. Bar, 10j.tm. lated with isoproterenol and isobutylmethylxanthine (Fig. 2B), demonstrating that changes in LD morphology during lipolysis A 120 were independent of the type of stimulus. As shown in a control ~ 100 •• c: • experiment with 100-nm fluorescent subresolution beads, LDs 80 • • of similar size should be clearly detectable with the microscope I ... 60 system used (Fig. 3B). This indicates that mLDs either do not ~ 40 . • :t: • #...... , occur or that their size is below the detection limit of the micro­ IV .r: 20 ... s'cope system used. Notably, no increase in fluorescence back­ 0 "~n r--r-r-rrTTrT l ground during lipolysis was observed, as would be expected 10 100 1000 upon the appearance of large numbers of small nano particles. log LD volume (tl) Interestingly, although cell volumes significantly shrunk over FIGURE 3.A, half-life of the volume of LOs upon lipolytic stimulation in 3T3-L 1 time during stimulated lipolysis, most LDs appeared to remain adipocytes. The volume of 46 selected LOs of an individual cell was calculated based on their diameter measured in maximum intensity projections of an at their relative position within the cell, suggesting that they are acquired four-dimensional data set. The half-life of the volume of LOs in stim­ associated with other subcellular structures (Fig. 2A). ulated cells appears to be dependent on the size of LOs. Larger LOs reach t'I, To test whether LD dynam­ more slowly than smaller LOs. B, eva luation of the microscope setup for mLDs Are Synthesized de Novo- detection of subresolution particles. 1OO -nm fluorescent microspheres were ics and morphology during lipolysis are influenced by the ability imaged with the same microscope setup as used for four-dimensional imag­ of cells to sequester excess FAs that derive from lipolysis, we ing of 3T3-L 1 cells. Groups of 100-nm beads and also individual particles are clearly detected. The image represents an enlarged area of a larger image. omitted BSA from the culture medium, which acts as FA scav­ Bar,1 j.tm. enger. In the absence ofBSA, existing LDs indeed decreased in size upon forskolin-stimulated lipolysis, indicating activation of the rapid formation of a large number of small LDs «1 J..lm) lipolysis (Fig. 4, A and B) . However, FA release from adipocytes within 30 min after initiation of lipolysis. These LDs grew sig­ was reduced by - 90%, compared with cells cultivated in the nificantly in size (- 1- 3 J..lm) within the following 60 -90 min presence of BSA (Fig. SA). Notably, the absence of BSA led to (Fig. 4, A - C) . Thus, mLD formation is strongly influenced by

11167 A +BSA -BSA o DMSO O DMSO • Trlacsin C + DMSO _ 50 • Triacsin C + DMSO 'iii 2500 CIl .. E 2000 E 40 «s= «s= :::: 1500 :::: 30 o o .[ 1000 .s.E 20 ~ 500 ~ 10 LL. LL. 0 ...... -- ...... "'-- o +-...... basal forskolln basal-"'-9-­ forskolin stimulated stimulated B 1. ~ 1.2 ~'iii 1.0 ~ .€ 0.8 1:'0 'iii E 0.6 s= I: y - o. CIl I: 0.2 FIGURE 4. Rapid formation of mLOs in 3T3-L 1 adipocytes after stimula­ .2 0~~~--4~~~~~~-~ tion of lipolysis in the absence of the extracellular fatty acid acceptor o 1 5 10 BSA, A, large numbers of small LDs «1 p.m in diameter) are detected 30 - 60 Triacsln C (j./molll) min after hormonal stimulation. B, lipolysis is not inhibited in the absence of BSA (arrows, enlarged areas of the white rectangular fields shown in A). Bar, 10 FIGURE 5. A, dependence of FA release on extracellular fatty acid acceptors in p.m. C, mLDs are formed in most cells of a cell population. Maximum intensity 3T3-L 1 adipocytes. There was a > 40-fold increase of FA release upon lipolytic projections are shown of three-dimensional data acquired at the indicated stimulation compared with basal conditions in cells containing DMSO and time points. Bar, 20 p.m. BSA. There was a - 6-fold increase of FA release upon lipolytic stimulation in the presence of triacsin C and BSA (left) . There were 80-fold (cells in DMSO) and SO-fold (cells in DMSO + triacsin C) decreases of FA release of cells cul­ the presence of the FA acceptor BSA in the culture medium tured without fatty acid acceptors (right) compared with cells cultured in the during stimulated lipolysis. These newly emerging mLDs were presence of BSA. Results are mean :':: S.D. (error bars) (three independent experiments). Statistical significance was determined by a two-tailed Stu­ not specifically detectable on the surface of existing LDs but dent's ttest (***, p < 0.001; **, p < 0.01). B, dose-dependent effect oftriacsin were rather dispersed throughout the cell. In addition, they did C on acyl-CoA levels in differentiated (day 8) adipocytes (see "Experimental not significantly change their relative subcellular position dur­ Procedures"). Maxi mal inhibition was achieved at 5 P.M triacsin C (--83%). Increasing concentrations of triac sin C (10 and 20 P.M) did not further reduce ing their growth (supplemental Movi e Sl). Furthermore, trans­ acyl-CoA levels. Results are mean' :':: S.D. (two independent experiments). mission electron micrographs acquired upon lipolytic stimula­ tion in the absence of BSA show a large number of mLDs (> 1 /Lm) but also of nLDs «l/Lm). Again, both nLDs and mLDs are not specifically detected on the surface of larger LDs but occur dispersed in the cell (Fig. 6). Taken together, these data suggest that formation of mLDs results from cellular FA overload in the absence of BSA in the media, which may trigger TAG (and LD) synthesis to prevent FA toxicity. Because FAs released from TAG by lipolytic deg­ radation require activation with coenzyme A prior to (re)incor­ poration into lipids, we next tested whether mLD formation during stimulated lipolysis is dependent on the activity of acyl­ CoA synthetases. For this purpose, we analyzed mLD formation FIGURE 6. Ultrastructural analysis of nLOs and mLOs formed upon lipo­ lytic stimulation in 3T3-L 1 adlpocytes Incubated In the absence of BSA, in the presence of triacsin C, an inhibitor of acyl-CoA synthe­ 3T3-L 1 adipocytes were treated with forskolin to stimulate lipolysis for 1 h in tase (24 -28). To evaluate the efficacy of triacsin C in cultured the absence of BSA. A sing le transmission electron microscopy section shows a larger number of dispersed nLDs and mLDs (left). Such small LDs are not adipocytes, we first determined the effect of the drug on acyl ­ detected on the surface of significantly larger LDs (> 10 p.m) (right) . Bar, 1 p.m. CoA levels in 8-daydifferentiated 3T3-Ll cell s. As shown in Fig. SB, triacsin C treatment of3T3-Ll cells led to a dose-dependent triacsin C, even in the absence of the FA acceptor BSA in the decrease in cellular acyl-CoA levels, indicative of efficient inhi­ medium. These data show that mLDs form de novo as a result of bition of ACS activity in vivo. Maximal inhibition was achieved cellular FA overflow during stimulated lipolysis and that TAG at S /LM triacsin C (- - 83%). Thus, the increase in FA release in degradation and new synthesis may operate in parallel to con­ response to triacsin C treatment as shown in Fig. SA correlates trol the level of (activated) FA in the cell. with a reduction in ACS activity. LDs Grow by Controlled Transfer of Lipids between As shown in Fig. 7, mLD formation was almost completely Organelles-The molecular mechanisms that govern LD inhibited during stimulated lipolysis in cell s treated with S JLM growth are currently unclear and may occur by spontaneous

11168 FIGURE 7. Inhibition of mLO formation in 3T3-L 1 adipocytes upon lipo­ FIGURE 8. Heterogeneity of LO size in differentiated adipocytes. Shown lytic stimulation in BSA-free medium using triacsin C. Celis degrade their are 3T3-L 1 celis (A) and human adipose-derived stem celis differentiated into lipid droplets to some extent (solid arrows) or contract without significant LD adipocytes (B) . Large LDs (arrows) are frequently located near the nucleus. consumption (open arrows). mLDs are not detected within 2 h of observation LDs of various sizes are scattered throughout the cytoplasm. CARS images 1 time. Bar, 10 /-Lm . Maximum intensity projections are shown of three-dim en­ were acquired at 2845 cm - (CH 2 symmetric stretching vibration). A maxi­ sional data acquired at the indicated time points. mum intensity projection is shown of acquired three-dimensional data. Bar, 10/-Lm. homotypic fusion of individual LOs (4), by selective lipid trans­ fer between LOs (19), or by TAG synthesis "on site" that is catalyzed by LO-resident acyltranferases (16). Subpopulations of LOs in differentiated adipocytes vary significantly in size; large LOs are frequently observed in the vicinity of the nucleus, whereas the smaller LOs form a size gradient toward the cellu­ lar periphery, both in murine 3T3-Ll cells (Fig. BA) and in human adipose-derived stem cells (Fig. BB). Because neutral lipid-specific -fluorescent dyes tend to promote LO fusion and influence intracellular LO movem ent upon microscopic inspection,S we applied label-free CARS microscopy for long term analysis of LO growth and assembly in living cells. To accelerate LO growth, 3T3-Ll cell s (day 7 after initiation of differentiation) were supplemented with oleic acid and ana­ lyzed by CARS microscopy for up to 16 h. As shown in Fig. 9 and FIGURE 9. Long term lipid transfer between LOs in 3T3-L 1 adipocytes. The large LD grows by complete "a bsorption" of a closely associated LD over supplemental Movie S2, several LOs grew by "absorbing" the hours. The lipid donor gets smalier over time (solid arrows), whereas the lipid lipid content of other, in all cases smaller, LOs. Importantly, acceptor grows over time (open arrows). Only sma Ii areas of the LD surfaces complete lipid transfer between larger "acceptor" LOs and are closely associated during this process. The participating LDs do not sig­ nificantly alter their rounded shape during lipid transfer. The process takes closely associated smaller "donor" LOs took up to several h, several h.lmaging was started 12 h after oleic acid treatment of the celis. CARS indicative of a finely tuned process rather than fast spontaneous images were acquired at 2845 cm - , (CH 2 symmetric stretching vibration). Maximum intensity projections are shown of three-dimensional data fusion. acquired at the indicated time points. Bar, 10 /-Lm. To test whether this mechanism of LO growth is induced by excess FA supply or is also occurring in untreated cells, we Notably, LO size distribution is typically rather heterogeneous analyzed LO dynamics in human adipose-derived stem cells in in adult adipocytes, both in cultured cell lines and in primary fat the absence of exogenous oleic acid. Similarly to the data cells, such as in murine white adipose tissue (29 -31). Currently, obtained with 3T3-Ll adipocytes in the presence of oleic acid, it is unclear how this heterogeneity is established and how the LOs grew by absorption of the neutral lipid content of smaller size distribution affects the metabolic fate of LOs. Using four­ LOs also in these cells and independent of exogenous FA sup­ dimensional live cell imaging, we have investigated in greater ply. Notably, individual LOs apparently took up the content of detail the dynamics and morphological alterations of LOs dur­ several other LOs, and acceptor LOs in turn served as lipid ing stimulated lipolysis as well as in m etabolically active but not donors for others. Again, the complete absorption process of proliferating cells. Virtually all LOs showed a progressive loss of individual LOs was not a spontaneous and rapid event but lipids independent of their size or subcellular position and were rather took place over a time period of several h. (Fig. 10 and almost totally depleted of lipids after 5 h of persistent stimula­ supplemental Movies S3 and S4). In summary, these observa­ tion. We conclude that under our experim ental conditions, lipo­ tions demonstrate that lipid transfer between LOs occurs in a lytic activity is not restricted to a subpopulation of LOs. Indeed, slow and regulated process without interaction of larger areas smaller LOs appeared to shrink faster than larger LOs, suggest­ of the LO surfaces. ing higher lipolytic activity on their surface, consistent with the increased surface/volume ratio and potentially increased den­ DISCUSSION sity of lipase molecules on their surface. Thus, the intracellular Understanding LO dynamics and is of great bio­ redistribution of neutral lipids and the tendency to generate medical interest in view of prevalent lipid-associated disorders. large LOs m ay function as an additional mechanism to regulate the rate of lipolysis. This strategy may be optimized in primary 5 H. Wolinski and C. Jungst, unpublished observations. adipocytes usually containing o ne very large and a number of

11169 - - - - - mentation nor the appearance of mLDs was observed, although .... • • cellular lipid stores were almost fully depleted in the course of •• -. •• •• • the experiment. On the other hand, 3T3-Ll cells rapidly pro­ Omin •• 30min •• 1h •• lh30min•• 2h •• duced a large number of mLDs upon stimulation of lipolysis if _

11170 appear to fuse but rather grew in size by de novo lipid deposi­ ing from the ER and/or LD fusion (57). Because LDs also harbor tion. Thus, we propose that fragmentation of larger LDs into diacylglycerol acyltransferase activity, it could be speculated nLDs and their subsequent fusion is not requir'ed to form mLDs that lipid transfer between individual droplets also occurs by a but rather resembles a mechanism compatible with de novo LD deacylation/FA activation/reacylation cycle. Such a mechanism formation at specific LD/ER contact sites (16). would explain the rather slow rate oflipid transfer between LDs Spatially highly resolved light microscopy of LD dynamics in and is compatible with the dual function ofRab18 GTPase both living cells is widely used due to the availability of intensely promoting LD fusion and lipolysis. However, because LD-asso­ emitting, lipophilic fluorophores, such as BODIPY 493/503 or ciated diacylglycerol acyltransferase 2 harbors a transmem­ LD540 (21). We have, however, observed that fluorescent label­ brane domain, it is controversial whether this enzyme directly ing may cause changes in the fusion behavior of LDs upon resides on the LDs or LD-associated subdomains of the ER (58) . (extended) light exposure.s To prevent such light-induced In conclusion, our findings suggest that formation of large alterations, we have employed CARS microscopy in this study LDs represents a regulated and slow physiological process in to assess long term LD dynamics. CARS is a nonlinear optical differentiating adipocytes, whereas mLDs form rapidly in process that allows label-fr ee monitoring of molecular vibra­ response to cellular FA overload and are synthesized to prevent tions and is therefore suited for the generation of molecule­ FA toxicity. We propose that LD growth requires a distinct specific contrast in unlabeled samples. CARS microscopy is protein machinery that mediates LD interactions, forms a highly effective for detection of lipids, due to the high density of channel between LDs, and thus promotes the transfer of lipids.

CH2 groups in lipid molecules (22, 50, 51). Optimized excita­ Indeed, elegant recent evidence demonstrates that Fsp27, a tion conditions allow CARS microscopic observations of indi­ member of the cell death-inducing DFF45-like effector (CIDE) vidual unstained cells over days without detectable cell damage family of proteins, localizes to the LD-LD interface and is (52). It should be noted that LDs of sizes below - 200 nm are not involved in mediating lipid transfer between adjacent lipid visible by CARS under the chosen excitation conditions. Thus, droplets in 3T3-Ll cells (59, 60). we cannot determine whether subresolution LDs grow by a process that is different from the observed long term lipid Acknowledgments-We thank Christina Eder and Viktor Adamek/or transfer between the fat storage organelles. technical assistance. Long term monitoring during adipocyte cultivation using CARS revealed a slow transfer of neutral lipids between closely associated LDs. 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