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Active involvement of micro-lipid droplets and lipid-droplet-associated proteins in hormone-stimulatedlipolysis in adipocytes

Takeshi Hashimoto1,2,*, Hiroki Segawa3, Masanari Okuno3, Hideaki Kano3,4, Hiro-o Hamaguchi3,Tokuko Haraguchi5,6, Yasushi Hiraoka5,6, Shiho Hasui2, Tomohiro Yamaguchi2,7, Fumiko Hirose2 andTakashi Osumi2,*1Faculty of Sport and Health Science, Ritsumeikan University, 1-1-1 Nojihigashi, Kusatsu, Shiga 525-8577, Japan2Graduate School of Life Science, University of Hyogo, 3-2-1 Koto, Kamigori, Hyogo 678-1297, Japan3Department of Chemistry, School of Science, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan4Institute of Applied Physics, University of Tsukuba, 1-1-1 Tennodai, Tsukuba, Ibaraki 305-8573, Japan5Graduate School of Frontier Biosciences, Osaka University, 1-3 Suita, Osaka 565-0871, Japan6Kobe Advanced ICT Research Center, National Institute of Information and Communication Technology, 588-2 Iwaoka, Nishi-ku, Kobe 651-2492,Japan7School of Pharmaceutical Science, Showa University, Tokyo 142-8555, Japan

*Authors for correspondence (thashimo@fc.ritsumei.ac.jp; osumi@sci.u-hyogo.ac.jp)

Accepted 15 October 2012Journal of Cell Science 125, 6127–6136� 2012. Published by The Company of Biologists Ltddoi: 10.1242/jcs.113084

SummaryThe regulation of lipolysis in adipocytes involves coordinated actions of many lipid droplet (LD)-associated proteins such as perilipin,hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), and its activator protein, CGI-58. Here, we describe the cellularorigin and physiological significance of micro LDs (mLDs) that emerge in the cytoplasm during active lipolysis, as well as the roles of

key lipolytic proteins on mLDs in differentiated 3T3-L1 adipocytes. Multiplex coherent anti-Stokes Raman scattering (CARS)microscopy demonstrated that mLDs receive the fatty acid (FA) moiety of triglyceride from pre-existing LDs during lipolysis. However,when FA re-esterification was blocked, mLDs did not emerge. Time-lapse imaging of GFP-tagged LD-associated proteins andimmunocytochemical analyses showed that particulate structures carrying LD-associated proteins emerged throughout the cells upon

lipolytic stimulation, but not when FA re-esterification was blocked. Overall lipolysis, as estimated by glycerol release, was significantlylowered by blocking re-esterification, whereas release of free FAs was enhanced. ATGL was co-immunoprecipitated with CGI-58 fromthe homogenates of lipolytically stimulated cells. Following CGI-58 knockdown or ATGL inhibition with bromoenol lactone, release of

both glycerol and FA was significantly lowered. AICAR, an activator of AMP-activated protein kinase, significantly increased FArelease, in accordance with increased expression of ATGL, even in the absence of CGI-58. These results suggest that, besides on thesurface of pre-existing central LDs, LD-associated proteins are actively involved in lipolysis on mLDs that are formed by FA re-

esterification. Regulation of mLDs and LD-associated proteins may be an attractive therapeutic target against lipid-associated metabolicdiseases.

Key words: Lipid droplet, Lipolysis, Lipid metabolism, Fatty acid, CARS microscopy

IntroductionExcessive lipid accumulation in adipocytes is a central feature of

obesity and metabolic syndrome. Lipid droplets (LDs) were long

regarded as metabolically inactive lipid storage depots lacking an

active role in cellular lipid metabolism. However, because of the

recent discovery of LD-associated proteins, they are now

considered as active organelles involved in diverse cellular

processes, such as membrane traffic and lipid metabolism (Farese

and Walther, 2009). LDs are composed of a central core of

triacylglycerol (TAG) or cholesterol ester (CE) and a surrounding

phospholipid monolayer. Excess energy is primarily stored as TAG

in LDs of adipose tissue, and the TAG reserves are hydrolyzed by a

process called lipolysis, to supply fatty acids (FAs) to various

tissues in cases of energy demand such as starvation and exercise.

The prevailing pathway of regulated lipolysis involving several

lipases and LD-associated proteins are summarized as follows

(Brasaemle, 2007; Granneman and Moore, 2008; Granneman et al.,

2009; Yamaguchi, 2010; Zechner et al., 2009). In quiescent

adipocytes, perilipin binds to comparative gene identification

(CGI)-58 (also called a,b-hydrolase domain-containing [ABHD]

5), a coactivator of adipose triacylglycerol lipase (ATGL), on the

surface of LDs, hence preventing CGI-58 from interacting with

ATGL. Perilipin also blocks hormone-sensitive lipase (HSL) from

accessing LDs. Upon lipolytic stimulation by catecholamines,

protein kinase A (PKA) phosphorylates perilipin, and

phosphorylated perilipin releases CGI-58. CGI-58 is now free to

interact with ATGL, and resulting ATGL/CGI-58 complex

efficiently hydrolyzes TAG to diacylglycerol (DAG) and FA.

DAG is then hydrolyzed to monoacylglycerol (MAG) and FA by

phosphorylated HSL that is now freed from blockage by perilipin to

be translocated to LD-surfaces. MAG is further decomposed to

glycerol and FA by MAG lipase.

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Using coherent anti-Stokes Raman Scattering (CARS)microscopy, we previously demonstrated that numerous micro-

lipid droplets (mLDs) appeared in all areas of the cytoplasm(Yamaguchi et al., 2007). We suggested that they are formed fromorganelles, e.g. endoplasmic reticulum (ER), but not from pre-

existing central LDs. A very recent study demonstrated, also byCARS microscopy, that mLDs are formed de novo during lipolysiswhen cellular FAs are overloaded, and suggested that mLDsprotect cells from the toxicity of excess FAs (Paar et al., 2012). On

the other hand, it was suggested that mLDs are derived from largecentral LD coated with perilipin, based on the observations thatmLDs are coated with perilipin and their formation required

phosphorylation of perilipin on Ser492 (Marcinkiewicz et al.,2006). It was also reported that LD-associated proteins includingATGL, HSL, perilipin and CGI-58 are distributed on small

particulate structures (Granneman et al., 2007). Hence, the cellularorigin and physiological significance of mLDs are to beestablished. It is also critical to discover where the LD-associated proteins functionally cooperate during active lipolysis,

given the observation that CGI-58 is dissociated from LDs inresponse to PKA activation (Yamaguchi et al., 2007). Finally, theroles of central LDs, mLDs, and other cellular structures in active

lipolysis should individually be defined.

In the present study, we performed microscopy and biochemicalstudies to examine the cellular origin and physiological

significance of mLDs as well as the functional interplay of LD-associated proteins during lipolysis in differentiated 3T3-L1adipocytes. We show that mLDs are formed by packaging of

TAG that was produced by FA re-esterification. They are engagedin active rehydrolysis of TAG to increase the proportion of FAs tobe released rather than re-stored.

ResultsTime-lapse imaging of LD-associated proteinsduring lipolysis

We first performed microscopy studies of the trafficking of key

lipolytic proteins in differentiated 3T3-L1 adipocytes. Duringlipolysis, GFP-perilipin was retained on the surface of large LDs,and also appeared on particulate structures in the cytoplasm

(Fig. 1A; supplementary material Movie 1). While some GFP-CGI-58 was also retained on the surface of large LDs, it wasdissociated from large LDs to the cytoplasm upon lipolyticstimulation, and appeared on particulate structures in the

cytoplasm (Fig. 1B; supplementary material Movie 2). On theother hand, both HSL and ATGL showed cytosolic distribution inthe basal state (Fig. 1C,D). During lipolysis, HSL was rapidly

redistributed to the surface of large LDs, and later exhibitedparticulate structures in the cytoplasm (Fig. 1C; supplementarymaterial Movie 3). While ATGL was mostly retained in the

cytoplasm during lipolysis, this protein was also redistributed tothe surface of large LDs, and exhibited particulate structures inthe cytoplasm, though the images were blurred by intense

cytosolic fluorescence (Fig. 1D; supplementary material Movie4). Overall, these lipid-associated proteins showed numerousparticulate structures in the cytoplasm of 3T3-L1 adipocytesupon lipolytic stimulation.

One could conceivably argue that the above dynamictrafficking of LD-associated proteins were an artifact observed

in the cells grown on two-dimensional (2D) flat surfaces. Suchcells are morphologically different from the unilocular fat cells in

vivo. We therefore cultured 3T3-L1 adipocytes in 3D collagen

matrix and examined the trafficking of GFP-perilipin upon lipolytic

stimulation (supplementary material Fig. S1). The mature

adipocytes contained 1–3 giant central LDs (.20 mm in diameter)

and peripheral small LDs in the cytoplasm in the basal state, and

GFP-perilipin was abundant in the peripheral LDs as previously

reported (Moore et al., 2005). In response to 1 hr lipolytic

stimulation, GFP-perilipin became observed on particulate

structures in the cytoplasm, besides retained on the surface of

large LDs. The appearance of these particulate structures was

similar to that in 2D-cultured cells, suggesting the physiological

relevance of the trafficking of LD-associated proteins.

Multiplex CARS imaging of mLDs during lipolysis

To examine where mLDs are derived from, we employed

multiplex CARS microscopy (for details, see Okuno et al.,

2010). We asked whether mLDs contain FAs derived from pre-

existing LDs or synthesized de novo. For this purpose, we pre-

loaded 3T3-L1 adipocytes with deuterium-labeled palmitate (D-

palmitate), and then subjected the cells to lipolysis in the absence

of D-palmitate (Fig. 2A). In the basal state, D-palmitate was

detected in the pre-existing large LDs (Fig. 2B). During lipolytic

stimulation, mLDs containing both C-H and C-D bonds appeared

in the cytoplasm, and increased with time (Fig. 2B). This result

suggests that mLDs receive FAs from pre-existing LDs, directly by

fragmentation of large LDs and/or indirectly by re-esterification of

FAs. The latter possibility seems particularly significant, because a

considerable portion of FA produced by lipolysis is usually re-

esterified in adipocytes (Reshef et al., 2003).

Hence, we next asked whether mLDs are formed as a

consequence of the fragmentation of large LDs or formed by

FA re-esterification. To address this issue, we inhibited the FA

Fig. 1. Live imaging of GFP-fused LD-associated proteins during

lipolysis. Differentiated 3T3-L1 cells were treated with 0.5 mM IBMX and

10 mM isoproterenol and time-lapse images of (A) GFP-perilipin, (B) GFP-

CGI-58, (C) GFP-HSL, and (D) GFP-ATGL during lipolysis were obtained

with a wide-field fluorescent microscope (DeltaVision). Arrows indicate

representative mLDs. Scale bars: 20 mm.

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re-esterification and TAG synthesis by an acyl-CoA synthetase(ACS) inhibitor, triacsin C, and a DAG acyltransferase (DGAT)

inhibitor, 2-bromooctanoate, and examined the appearance ofmLDs by multiplex CARS microscopy. mLDs did not appeareven after 32 min or 64 min of lipolytic stimulation, when re-

esterification of FAs was inhibited (Fig. 2C,D). This resultindicates that mLDs are formed by re-esterification of FAsderived from central LDs and possibly in part from the media.

Distribution of LD-associated proteins under inhibition ofFA re-esterification

We examined whether the particulate structures containingperilipin, CGI-58, HSL, and ATGL were generated duringlipolysis, when FA re-esterification was inhibited. In the basal

state, similar to the results on GFP-tagged proteins (Fig. 1), the

localization of HSL was cytoplasmic, whereas that of perilipin was

on large central LDs (Fig. 3A). Upon lipolytic stimulation, HSL

was redistributed to the surface of large central LDs and coexisted

with perilipin. Furthermore, HSL and perilipin were distributed on

small particulate structures throughout the cytoplasm. When re-

esterification of FAs was inhibited, the structures coated with both

HSL and perilipin did not emerge, both proteins coexisting solely

on the surface of large central LDs. This distribution pattern lasted

even during 6 hrs after lipolytic stimulation. These results indicate

that the microstructures carrying perilipin and HSL are consistent

with and likely to be the mLDs that are visualized by CARS

microscopy.

Distribution of CGI-58 and ATGL during lipolysis was

examined under the same conditions (Fig. 3B). In the basal state,

the localization of ATGL was cytoplasmic, while that of CGI-58

was on large LDs. Upon lipolytic stimulation, CGI-58 was

translocated to the cytoplasm with a speckle-like pattern, where

being co-localized at least partially with ATGL. Even when re-

esterification of FAs was inhibited, CGI-58 was translocated to the

cytoplasm with a speckle-like pattern in response to lipolytic

stimulation, being co-localized at least partially with ATGL,

whose cytoplasmic distribution was not altered during lipolysis as

in Fig. 1D and supplementary material Movie 4.

On the other hand, when cells were permeabilized with 0.01%

digitonin before fixation with paraformaldehyde to decrease the

cytosolic staining (see Materials and Methods), CGI-58 was

retained on large LDs, while ATGL was depleted in the basal

state (Fig. 3C). Upon lipolytic stimulation, ATGL was observed on

Fig. 2. Imaging of mLDs prelabeled with deuterated palmitate to

investigate the origin of TAG contained in mLDs during lipolysis by

multiplex CARS microspectroscopy. (A) This technique reveals the

distributions of different chemical bonds, such as C-D and C-H bonds, at the

same time in living cells. Deuterium-labeled palmitate was added to the

medium and allowed to be incorporated into large LDs of differentiated 3T3-

L1 adipocytes for 24 hrs. It was examined whether newly appearing mLDs

during lipolysis contain C-H bond (gray particles: nascent mLDs formed by

esterifying FAs synthesized de novo) or C-D bond (orange particles: products

of the fragmentation of pre-existing large LDs or FA re-esterification), or

both, by multiplex CARS microscopy. (B) Differentiated 3T3-L1 cells were

treated with 0.5 mM IBMX and 10 mM isoproterenol for up to 80 min and the

CARS spectra of C-H bonds (stretching vibration at 2845 cm21) or C-D

bonds (stretching vibration at 2090 cm21) were detected every 8 min by

multiplex CARS microspectroscopy. Images at the basal state, and 8 min,

32 min, 56 min, and 80 min after lipolytic stimulation are shown. Alterations

in mLD number were examined by two independent observers, and their

counts were averaged. The number of mLDs containing C-H bonds was 45,

64, 95, 129, and 159 at the basal state and 8 min, 32 min, 56 min, and 80 min

after lipolytic stimulation, respectively. The number of mLDs containing C-D

bonds was 22, 41, 67, 98, and 130 at the basal state, and 8 min, 32 min,

56 min, and 80 min after lipolytic stimulation, respectively. Note that some

H-labeled mLDs may be judged negative for D-label because of the lower

contrast of C-D bond images than those of C-H bonds. Each panel is by

30 mm630 mm. Arrows indicate representative mLDs. (C) Multiplex CARS

microscopic images of C-H bonds during lipolysis under inhibition of FA re-

esterification. Even after 32 or 64 min of lipolytic stimulation, mLDs did not

appear when re-esterification of FAs was inhibited with triacsin C and 2-

bromooctanoate. The number of mLDs containing C-H bonds was 3, 3, and 2

at the basal state and 32 min, and 64 min after lipolytic stimulation,

respectively. (D) A larger field view of multiplex CARS microscopic images

of C-H bonds during lipolysis under inhibition of FA re-esterification is

shown. mLDs did not appear in any cell when re-esterification of FAs was

inhibited with triacsin C and 2-bromooctanoate during 2 hrs of lipolysis.

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the surface of central large LDs where being co-localized with CGI-

58. Furthermore, ATGL and CGI-58 were co-localized at particulate

structures. When re-esterification of FAs was inhibited, these

particles mostly disappeared. This result suggests that ATGL and

CGI-58 are also co-localized on the surface of large LDs as well as

on particulate structures, probably mLDs, during lipolysis. Similar

distribution patterns were observed in double-immunostaining with

the combinations of perilipin-HSL, perilipin-ATGL, and CGI-58-

HSL (Fig. 3C).

Previously, Yamaguchi et al. observed that mLDs emerged

upon lipolytic stimulation in 3T3-L1 cells in which CGI-58 was

knocked down with siRNA (Yamaguchi et al., 2007), and hence

suggested that CGI-58 is not required for the formation of mLDs.

In accordance with this idea, we also observed that the particulate

distribution of HSL and ATGL in the CGI-58 knockdown cells

responding to lipolytic stimulation was not different from that in

normal 3T3-L1 cells (data not shown).

A previous study showed that mLDs are not formed in

lipolytically stimulated 3T3-L1 adipocytes, when 2% BSA (Paar

et al., 2012) was added to the culture medium. We found,

however, that the particulate structures containing perilipin and

HSL appeared upon lipolytic stimulation even in the presence of

2% BSA (data not shown). Again, when re-esterification of FAs

was inhibited, these particulate structures did not appear.

Although the reason for the inconsistency between the previous

and present studies is not clear, a possibility is that the lipolytic

stimulation in the present study (0.5 mM IBMX and 10 mM

isoproterenol) was strong enough to induce overflow of cellular

FA.

Lipolytic activity under inhibition of mLD generation

Next, we explored the significance of mLDs in lipolysis. To

compare lipolytic activities in the presence and absence of mLDs,

release of FA and glycerol into the media during lipolytic

stimulation was measured with (2Br-TriaC) or without (Control)

Fig. 3. Distribution of endogenous perilipin, CGI-58, HSL, and ATGL

during lipolysis in 3T3-L1 adipocytes. (A) Differentiated 3T3-L1 cells were

treated with 0.5 mM IBMX and 10 mM isoproterenol for the time indicated

and permeabilized with 0.2% Triton X-100/PBS after fixation with

paraformaldehyde, and distribution of endogenous HSL (green) and perilipin

(red) during lipolysis was obtained by immunofluorescence microscopy.

(A-a) In the basal state, the localization of HSL was cytoplasmic, while that

of perilipin was on large LDs. (A-b) Upon lipolytic stimulation, HSL was

redistributed to the surface of large central LDs and coexisted with perilipin.

Furthermore, HSL and perilipin were distributed on small particulate

structures throughout the cytoplasm. (A-c) When re-esterification of FAs was

inhibited by the treatment of triacsin C and 2-bromooctanoate, mLDs coated

with both HSL and perilipin did not appear, although HSL and perilipin were

co-localized on the surface of large LDs. (A-d) Under inhibition of FA re-

esterification, HSL/perilipin-coated mLDs did not appear even 6 hrs after

lipolytic stimulation. Arrows indicate representative of mLDs. Scale bar:

20 mm. (B) Differentiated 3T3-L1 cells were treated with 0.5 mM IBMX and

10 mM isoproterenol for the time indicated and permeabilized with 0.2%

Triton X-100/PBS after fixation with paraformaldehyde, and distribution of

Myc-CGI-58 (green) and ATGL (red) during lipolysis was obtained by

immunofluorescence microscopy. (B-a) In the basal state, ATGL was located

throughout the cytoplasm, while CGI-58 was on large LDs. (B-b) Upon

lipolytic stimulation, CGI-58 was translocated to the cytoplasm with a

speckle-like pattern, where being co-localized at least partially with ATGL.

(B-c) Even when re-esterification of FAs was inhibited with triacsin C and 2-

bromooctanoate, CGI-58 was translocated to the cytoplasm and co-localized

with ATGL. (B-d) A similar pattern of distribution was observed after 6 hrs

of lipolytic stimulation in the presence of triacsin C and 2-bromooctanoate.

Bar, 20 mm. (C) Differentiated 3T3-L1 cells were treated with 0.5 mM IBMX

and 10 mM isoproterenol for 1 hr and permeabilized with 0.01% digitonin

before fixation with paraformaldehyde, and lipid-associated proteins were

detected by confocal microscopy. (C-a) In the basal state, perilipin (red) and

CGI-58 (red) were found on large LDs, while HSL (green) and ATGL (green)

were not detected. (C-b) Upon lipolytic stimulation, HSL (green) and ATGL

(green) were distributed on the surface of large central LDs and coexisted

with either perilipin (red) or CGI-58 (red). Furthermore, these proteins were

also distributed on small particulate structures throughout the cytoplasm.

(C-c) Note that when re-esterification of FAs was inhibited by the treatment

with triacsin C and 2-bromooctanoate, microstructures coated with perilipin,

CGI-58, HSL and ATGL did not appear, while the pairs of perilipin-HSL,

perilipin-ATGL, CGI-58-HSL, and CGI-58-ATGL were co-localized on the

surface of large LDs. Arrows indicate representative mLDs. Scale bar: 20 mm.

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2-bromooctanoate and triacsin C (Fig. 4). As expected, FA release

was significantly increased with the inhibitors (2Br-TriaC) as

compared with Control, indicating that FAs are in a large part re-

esterified under usual lipolytic conditions, which were released

into media when re-esterification was blocked (Fig. 4A).

Consistent with this, total TAG levels and the size of large

central LDs were reduced to a larger extent in the presence of the

inhibitors of FAs re-esterification than those under usual lipolytic

conditions (supplementary material Fig. S2). On the other hand,

the inhibitors significantly reduced glycerol release (Fig. 4B).

Adipocytes have poor activity of glycerol kinase that is essential

for the production of glycerol 3-phosphate, a key substrate for

TAG synthesis (Leroyer et al., 2006; Reshef et al., 2003). Thus,

glycerol produced by lipolysis in adipocytes is not efficiently

reused as the partner of FA re-esterification, but mostly released

from the cells immediately. Hence, the glycerol moiety of TAG

stored in mLDs, and released from there by lipolysis, is

continuously supplied de novo (mostly by glycolysis in the

present experimental setting), but not by lipolysis at central LDs.

In Control, glycerol release represents the sum of lipolytic

activities at central LDs and mLDs, whereas in 2Br-TriaC, only

lipolysis at central LDs because of the absence of mLDs. Thus, the

decrease in glycerol release in 2Br-TriaC as compared with that in

Control most conceivably represents the loss of lipolysis at mLDs,

suggesting the significant contribution of mLDs to active lipolysis.

As both central LDs and mLDs release FAs by lipolysis, assuming

a similar relative proportion of those FAs from either pool being

re-esterified, the contribution of mLDs to FA release would be

estimated (mLDs contribution); this would be achieved by

applying the fraction for relative contribution of mLDs in

glycerol release to FA release from Control cells (Fig. 4A,

dotted line). Thus, mLDs contributes to nearly 50% of total FA

release during lipolysis, indicating that a large amount of FAsgenerated by lipolysis is re-esterified into TAG, which is activelyre-hydrolyzed on mLDs. Additionally, even in DMEM/10% FBS,

glycerol release was significantly decreased, whereas FA releasewas significantly increased with the inhibitors (2Br-TriaC) (datanot shown). These results mean that mLDs are involved in activelipolysis, contributing to increase in FA release.

Subcellular distribution and interaction of CGI-58and ATGL

To exactly locate where CGI-58 and ATGL interacts, we analyzed

their intracellular distribution before and after lipolytic stimulationby subcellular fractionation (Fig. 5A). In the basal state, ATGLwas mainly cytosolic (Cyt), and also found in LD (LD) and pellet

(Pel) fractions. On the other hand, CGI-58 was mainly found in theLD fraction. Upon lipolytic stimulation, the contents of ATGL inthe LD fraction and CGI-58 in the Pel fraction increased, where

ATGL and CGI-58 now abundantly co-existed.

We next examined the association of CGI-58 and ATGL

in response to lipolytic stimulation. Immunoprecipitation ofexogenously expressed Flag-CGI-58 was performed in the lysatesfrom 3T3-L1 adipocytes that were lipolytically stimulated or not(Fig. 5B). Endogenous ATGL was co-immunoprecipitated with

Flag-CGI-58 upon 2 hrs of lipolytic stimulation.

Significance of ATGL and CGI-58 in lipolysis

Given with the interaction of CGI-58 and ATGL upon lipolytic

stimulation, release of glycerol and FAs was measured duringlipolytic stimulation, in the presence (BEL (+)) or absence (BEL

(2)) of bromoenol lactone (BEL), an irreversible inhibitor of the

iPLA2 family of phospholipases/lipases including ATGL.

Fig. 4. Lipolytic activity under inhibition of the generation of mLDs.

Release of (A) FAs and (B) glycerol into the medium with (2-Br-TriaC, solid

line, closed circles) or without (Control, dashed line, open circles) 2-

bromooctanoate and triacsin C were measured in differentiated 3T3-L1 cells

in the presence of 0.5 mM IBMX and 10 mM isoproterenol. In A, the

estimated contribution of mLDs to FA release is also shown (mLDs

contribution, dotted line, open squares). **P,0.01 versus 2Br-triaC.

Fig. 5. Co-localization and interaction of CGI-58 and ATGL during

lipolysis. (A) Subcellular fractionation on CGI-58 and ATGL before and after

lipolytic stimulation. Differentiated 3T3-L1 cells were treated with or without

0.5 mM IBMX and 10 mM isoproterenol for 60 min, and separated into lipid

droplet (LD), cytosolic (Cyt), and pellet (Pel) fractions (see Materials and

Methods). CGI-58 and ATGL in each fraction was analyzed by western

blotting. Perilipin was predominantly found in the LD fraction, suggesting

that the subcellular fractionation was successful. Note that the

phosphorylation of perilipin by PKA was revealed as a shift of electrophoretic

mobility by immunoblotting. (B) Interaction of CGI-58 and ATGL during

lipolysis in 3T3-L1 adipocytes expressing Flag-tagged CGI-58.

Immunoprecipitation of exogenously expressed Flag-CGI-58 was performed

for the lysates (Input) of 3T3-L1 adipocytes with or without 2 hrs lipolytic

stimulation, and endogenous ATGL in the immunoprecipitate (IP) was

analyzed. Normal IgG was used as a nonspecific control. Immunoblots (IB) of

ATGL and CGI-58 are shown.

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Experiments were performed with 3T3-L1 adipocytes treated

(2Br-triaC) or not (Control) with 2-bromooctanoate and triacsin

C (Fig. 6A,B). BEL reduced the activity of PKA-stimulated

lipolysis in both Control and 2-Br-TriaC conditions, suggesting

that ATGL is indeed involved in active lipolysis.

We also examined the significance of CGI-58 in lipolysis with

3T3-L1 adipocytes in which CGI-58 was knocked down with

siRNA (CGI-58 KD). Glycerol release in response to lipolytic

stimulation was significantly decreased in CGI-58 KD cells as

compared to that in control cells (mismatch) (P,0.01) (Fig. 6C),

consistent with our previous report (Yamaguchi et al., 2007).

Notably, FA release was dramatically more decreased in CGI-58

KD cells as compared to that in mismatch cells (P,0.01)

(Fig. 6D,E). When 3T3-L1 adipocytes were treated with 2-

bromooctanoate and triacsin C (2Br-triaC), CGI-58 knockdown

had little effect on glycerol or FA release as compared to

mismatch control cells.

We examined whether the apparent effect of CGI-58 RNAi

was exactly due to knockdown of CGI-58 itself, not an off-target.

For this purpose, a rescue experiment was performed by

expressing a mutant CGI-58 mRNA that is not targeted withthe siRNA, still encoding the wild-type amino acid sequence, inthe CGI-58 KD cells (supplementary material Fig. S3). The

rescued cells exhibited significant increase in the release of bothglycerol and FA, and importantly, more prominently in FArelease. This result supports the specificity of CGI-58 knockdown

in the present study (Fig. 6F,G).

Effects of AICAR on lipolytic activity and the traffic of LD-associated proteins

Previously, Gaidhu et al. demonstrated that activation of AMP-activated protein kinase (AMPK) with N1-(b-D-Ribofuranosyl)-5-

aminoimidazole-4-carboxamide (AICAR) increased ATGLactivity and FA release in response to lipolytic stimulation(Gaidhu et al., 2009). However, AICAR inhibited HSL activity,

glycerol release upon lipolytic stimulation, and incorporation ofglucose, FA, and glycerol into lipids. Accordingly, the authorssuggested that FA re-esterification and lipogenesis were reduced

by AICAR treatment (Gaidhu et al., 2009). Their suggestionprompted us to examine whether AICAR treatment decreased thegeneration of mLDs, thereby increasing FA release whereas

decreasing glycerol release in response to lipolytic stimulation. Wefound that AICAR treatment significantly increased FA release,whereas it did not affect glycerol release (Fig. 7A). Furthermore,emergence of mLDs as assessed by immunostaining of perilipin,

HSL, CGI-58, and ATGL was not influenced by AICAR treatment(data not shown). AICAR treatment significantly increased theamounts of phospho-acetyl-CoA carboxylase (Ser79) (p-ACC),

phospho-AMPK (Thr172) (p-AMPK), and ATGL. No change,however, was observed in the amounts of perilipin, phospho-HSL(Ser565) (p-HSL), HSL, DGAT1, and CGI-58 (Fig. 7B,C).

Finally, we examined whether the effects of AICAR treatment

are still observed even when CGI-58 was knocked down. AICARtreatment significantly increased FA release, although no changewas observed in glycerol release, in CGI-58-KD cells(supplementary material Fig. S4). We also found that AICAR

treatment significantly increased the amounts of p-ACC, p-AMPK,and ATGL, whereas no change was observed in perilipin, p-HSL,and HSL in these cells (supplementary material Figs S5, S6).

DiscussionThe present study provides important findings in understandingmolecular mechanisms of lipolysis in adipocytes: during lipolysis,significant amount of FAs generated by TAG hydrolysis are re-

esterified, leading to formation of mLDs. These mLDs are coatedwith perilipin, HSL, CGI-58, and ATGL, representing active sitesof lipolysis.

mLDs were already noted by other researchers, and purportedto be derived from central large LDs (Brasaemle et al., 2004; Guo

et al., 2008; Marcinkiewicz et al., 2006; Miyoshi et al., 2007). Onthe other hand, a very recent study revealed, by using real-timequantitative imaging and electron tomography, that mLDs are

formed throughout the cytoplasm during lipolysis, but are notformed by the fission from large LDs (Ariotti et al., 2012). Inaddition, another very recent study revealed, by using CARS

microscopy, that mLDs are formed de novo during lipolysis whenexcess FAs are accumulated in the cells (Paar et al., 2012). In thepresent study, we used multiplex CARS microspectroscopy

(CARS spectral imaging method) in order to elucidate the originof the TAG contents in mLDs. CARS microscopy has highsensitivity to lipids abundant in long-chain hydrocarbons by

Fig. 6. Effects of bromoenol lactone (BEL), an irreversible ATGL

inhibitor, and CGI-58 knockdown on lipolysis. (A) Glycerol release and

(B) FA release were measured during lipolytic stimulation, with (BEL (+),

filled bar) or without (BEL (2), open bar) in 3T3-L1 adipocytes, treated with

(2Br-triaC) or without (Control) 2-bromooctanoate and triacsin C.

(C–E) 3T3-L1 adipocytes in which CGI-58 was knocked down (filled bar)

and that of mismatch control cells (open bar) were differentiated. Cells were

analyzed for (C) glycerol release, (D) FA release, and (E) the ratio of FA/

glycerol release in response to lipolytic stimulation under conditions in which

FA re-esterification was inhibited (2-Br-TriaC) or not (Control). (F) Glycerol

and (G) FA release of CGI-58-KD cells expressing siRNA-resistant CGI-58

mutant (Mutant) and empty vector control (Empty). (A,B) **P,0.01 versus

BEL (2); ##P,0.01 versus Control; (C–G) **P,0.01 versus mismatch or

Empty control; ##P,0.01 versus Control.

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tuning the laser wavelengths to CH stretch vibrational mode, and

has proven to be an excellent tool for LD tracking (for a review,

see Evans and Xie, 2008). The deuterium in a CD bond is heavier

than hydrogen, placing CD vibrational stretching frequencies

into near 2100 cm21, which is an otherwise silent region of

the biological Raman spectra. By incubating differentiated

adipocytes with deuterium-labeled palmitate and replacing

some CH bonds with CD bonds, it is possible to selectively

image a specific chemical bond (i.e. CD bond) using CARS

contrast. The multiplex CARS microspectroscopy enabled us to

obtain simultaneous imaging of CD and CH stretch vibrational

modes, and revealed that newly appearing mLDs are formed as a

consequence of the fragmentation of large LDs and/or the re-

esterification of intracellular FAs. On the other hand, with ACS

and DGAT inhibitors, emergence of mLDs was suppressed.

These results indicate that mLDs are formed by re-esterification

of FAs derived from central LDs, presumably occurring at the

ER, because ACS and DGAT are localized primarily in ER, and

ER membranes are often found close to LDs (Blanchette-Mackie

et al., 1995; Brasaemle and Wolins, 2012). Although the precise

mechanism that newly synthesized TAG is packaged in mLDs is

uncertain, the prevailing model postulates that TAG is

accumulated between the leaflets of phospholipid bilayer, and

resulting lens-shaped structures are then pinched off (Brasaemle,

2011; Brasaemle and Wolins, 2012; Farese and Walther, 2009). It

seems reasonable that mLDs are formed at ER, because ER can

serve to provide adequate amounts of phospholipid to cover the

large surface areas of the newly synthesized mLDs (Farese and

Walther, 2009; Guo et al., 2008; Ohsaki et al., 2009).

Next, we examined the significance of mLDs as well as the

functional interplay of LD-associated proteins in lipolysis. The

significance of mLDs might be explained by the increase in the

surface area available for lipolysis. Indeed, by a genome-wide

RNAi screen in Drosophila Schneider 2 cells, Guo et al.

demonstrated a defect in lipolysis in cells containing only a

few large central LDs due to the lack of Cct1, which encodes an

enzyme catalyzing the rate-limiting step in phosphatidylcholine

synthesis (Guo et al., 2008). In addition, a previous study

suggested a higher lipolytic activity on mLDs by the observation

that smaller LDs shrunk faster than larger LDs (Paar et al., 2012).

In the present study, overall lipolysis as estimated by glycerol

release was significantly lowered by blocking FA re-

esterification and thus mLD formation. During TAG synthesis

on mLDs, glycerol produced by lipolysis was virtually all derived

from glucose through glycolysis, not from free glycerol (Fig. 8);

therefore, it is likely that the lowered glycerol release by re-

esterification inhibitors represents loss of contribution of mLDs

in total lipolysis rather than any change in intracellular utilization

of free glycerol (see Results). Thus, mLDs are most probably

active sites of lipolysis. In accordance with this, HSL and ATGL,

pivotal lipases in adipocytes, co-existed with perilipin and CGI-

58 on mLDs, which disappeared upon blocking FA re-

esterification. The present study shows substantial rates of FA

re-esterification, implicating a ‘‘futile metabolic cycle’’. What is

the significance of mLDs generated on the basis of a futile

metabolic cycle? The net efflux of FAs from the adipocytes,

Fig. 7. Effect of AICAR on lipolytic activity. (A) Release of glycerol and

FA. (B) Estimation by densitometric scan of immunoblots for p-ACC, p-HSL,

HSL, p-AMPK, perilipin, ATGL, CGI-58, and DGAT1, normalized with

GAPDH. (C) Representative immunoblots examining the effect of AICAR

treatment. Extracts from 3T3-L1 cells treated with (AICAR) or without

(Control) AICAR were immunoblotted for p-ACC, p-HSL, HSL, p-AMPK,

perilipin, ATGL, CGI-58, DGAT1, and GAPDH. *P,0.05, and **P,0.01

versus Control.

Fig. 8. Model of active lipolysis in adipocytes. Significant amounts of FAs

produced by TAG hydrolysis are re-esterified into TAG, leading to the

formation of mLDs at the endoplasmic reticulum (ER) even during active

lipolysis. mLDs are coated with perilipin (peri), HSL, CGI-58 (CGI) and

ATGL, and are active sites of lipolysis. CGI-58 and ATGL may also co-

operate on the ER. G3P, glycerol 3-phosphate.

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determined by the balance between lipolysis and FA re-esterification, can at most be equal to the total lipolytic rate

and is typically less because of an active FA re-esterificationprocess. Increased lipolysis assisted by mLDs would be asignificant way to enhance the net efflux of FAs even without achange in re-esterification rate. The fractional turnover rate of

mLDs is much higher than that of large LDs, so a relative shift inthe site of TAG turnover from large LDs to mLDs would greatlyincrease the probability of re-stored FAs being rapidly re-

lipolyzed and thus mobilized from the cells. Overall, theappearance of rapidly turning-over mLDs may have aphysiological relevance to enhance the lipolytic activity,

thereby preventing an abundance of FAs recycling back toslowly turning-over pools of glycerolipids and phospholipids.Additionally, if there is any means of direct transfer of TAG fromcentral LDs to mLDs, not via hydrolysis and re-esterification,

mLDs would also be able to support the increase in net lipolyticcapacity. Protein-mediated transfer or vesicular transport wouldbe the possible means, though neither of them has been identified

as a mechanism of TAG transfer.

Again, the present study showed the elaborated cooperation ofperilipin, CGI-58, HSL, and ATGL on mLDs during lipolysis. In

addition to previously reported functional interaction of HSL withperilipin on LDs (Granneman et al., 2007; Miyoshi et al., 2006; Shenet al., 2009), interaction of exogenously expressed CGI-58 andATGL on the surface of LDs was also confirmed by biomolecular

fluorescence complementation (BiFC) or co-immunoprecipitationstudies in COS7 and CHO-K1 cells (Granneman et al., 2009; Wanget al., 2011). In the present study, we demonstrated in differentiated

3T3-L1 adipocytes that endogenous CGI-58 and ATGL are co-localized on the surface of LDs including mLDs, and Flag-taggedCGI-58 expressed at a physiological level is co-immunoprecipitable

with ATGL upon lipolytic stimulation. Furthermore, the presentstudy demonstrates that BEL, an irreversible inhibitor of the iPLA2family of phospholipases/lipases including ATGL, reduced the

activity of PKA-stimulated lipolysis. These results suggest thatATGL is involved in active lipolysis interacting with CGI-58, atleast in part on the surface of LDs including mLDs. This findingcorroborates the previous report showing in a fluorescence

resonance energy transfer (FRET) experiment that CGI-58 andATGL exhibit interaction on particulate structures upon PKAactivation (Granneman et al., 2007).

We also observed in immunocytochemical analysis that uponlipolytic stimulation, CGI-58 is translocated to the cytoplasmwith a speckle-like pattern, where being co-localized at least

partially with ATGL. In addition, a subcellular fractionationstudy indicated that ATGL and CGI-58 co-existed in not only LDfraction, but also cytosolic and pellet fractions, the latter of whichcontained membranous organelles. Although the precise entity

and physiological significance of their cytoplasmic locationremain to be elucidated, previous reports suggested that CGI-58is a coenzyme A-dependent lysophosphatidic acid acyltransferase

(LPAAT) (Ghosh et al., 2008; Montero-Moran et al., 2010), andLPAAT enzymes are located in both ER and Golgi complex(Schmidt and Brown, 2009). Hence, CGI-58 may act as LPAAT

at organelles such as ER and Golgi. On the other hand, we foundin the present study that CGI-58-KD cells suffered a drasticdecrease in FA release as compared with the reduction in the

glycerol release, suggesting an unidentified role of CGI-58 topromote FA release. The enhanced decrease in FA release ascompared with glycerol release by CGI-58 knockdown was

diminished, if FA re-esterification was inhibited and thus mLDswere not generated. Thus, we conclude that the lipolytic function

of CGI-58 is predominant on the surface of mLDs, and is relatedto recruiting FA to be secreted rather than re-esterified.

AMPK is a sensor of cellular energy state that responds to

metabolic stress and other regulatory signals, and theadministration of AMPK activator AICAR to rats diminishedadiposity (Ruderman et al., 2003b). Given the previous study

suggesting the effect of AICAR on reduced re-esterification of FAand lipogenesis (Gaidhu et al., 2009), we hypothesized thatAICAR treatment may have a similar effect to that by the

inhibition of FA re-esterification with 2-bromooctanoate andtriacsin C. In the present study, we found that AICAR treatmentsignificantly increased FA release, but without decreasing glycerolrelease, contrary to the previous observation (Gaidhu et al., 2009).

As the appearance of mLDs decorated by perilipin, HSL, CGI-58,and ATGL was not influenced by AICAR treatment, it is notsurprising that the glycerol release, representing the total lipolytic

activity, was not decreased. Because AICAR treatmentsignificantly increased the expression of ATGL, and its co-activator CGI-58 seemed to have a role to increase FA release,

ATGL may also increase the ratio of FAs to be released. Hence,increasing the AMPK activity in adipose tissue, for instance byendurance exercise (Ruderman et al., 2003a), could be an attractivetarget against obesity-associated metabolic disorders.

In summary, we conclude that significant amount of FAsproduced by TAG hydrolysis are re-esterified into TAG at ER

during lipolysis to form mLDs (Fig. 8). These mLDs are coatedwith perilipin, HSL, CGI-58 and ATGL, and are active sites oflipolysis, probably devoted to increasing total TAG turnover andthus potentially net FA release (Fig. 8). CGI-58 is also possibly

dispersed to structures including organelles such as ER and/orGolgi apparatus upon lipolytic stimulation, the physiologicalmeaning of which is to be elucidated.

Materials and MethodsConstruction of recombinant lentiviruses

cDNAs of mouse CGI-58, HSL and ATGL were obtained as described previously(Yamaguchi et al., 2004; Yamaguchi et al., 2007). Mouse perilipin cDNA wasobtained from adipocyte total RNA by reverse transcription (RT)-PCR. GFP-fusionconstructs were prepared using phGFP(105), a GFP mutant that has an increasedbrightness (Yamasaki et al., 1999). From a lentiviral expression vector, CSII-hMTIIA(DGRE)-MCS-IRES2-Venus (Fumoto et al., 2007), the IRES2-Venusregion was removed using appropriate restriction sites, and GFP(105)-codingsequence was inserted into the multicloning site, yielding CSII-MT-GFP. cDNAs ofthe LD-associated proteins were then inserted into CSII-MT-GFP. cDNA of CGI-58was also subcloned into CSII-MT-myc or CSII-MT-Flag plasmid, in which theGFP(105)-coding sequence was replaced with that of myc or Flag tag.

Recombinant lentiviruses were prepared with these plasmid constructs as describedpreviously (Yamashita et al., 2007; Yamashita et al., 2004). A lentivirus vectorexpressing short hairpin RNA (shRNA) against mouse CGI-58 was prepared asreported previously (Yamaguchi et al., 2007). For a rescue experiment, a mutant CGI-58 cDNA in which the siRNA target sequence, 59-AAGAAGTAGTAGACCT-AGGTT-39, was changed to 59-AAGAAGTAGTCGTCCACGTTT-39, but stillencoding the wild-type amino acid sequence, was inserted in CSII-MT-flag vectorcontaining a flag tag sequence instead of myc.

Cell culture

All reagents for cell culture were obtained from Wako (Osaka, Japan) unlessotherwise mentioned. 3T3-L1 cells were cultured and differentiated as describedpreviously (Yamaguchi et al., 2007). Three-dimensional culture of 3T3-L1adipocytes was prepared as follows. Differentiated adipocytes from one 6-cm dishwere trypsinized and resuspended in the mixture of ice-cold Cellmatrix Type I-A(Nitta Gelatin Inc., Osaka, Japan), DMEM, 50 mM NaOH, 260 mM NaHCO3, and200 mM HEPES. Suspension of 26105 cells was placed in a 35-mm glass bottomdish and allowed to solidify at 37 C for 30 min. Growth medium (DMEM/10%FBS (Nichirei Biosciences, Tokyo, Japan) supplemented with 5 mg/ml insulin) wasadded, and the cells were maintained for two weeks. In some experiments, AICAR

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(Sigma-Aldrich, St. Louis, MO, USA) was added to differentiated 3T3-L1adipocytes at 2 mM for 12 hrs prior to the assay for lipolytic activity and westernblotting.

Lipolytic stimulation

For microscopy studies, lipolytic stimulation was applied to differentiated 3T3-L1adipocytes as follows: After washing twice with Hank’s buffer, cells wereincubated with DMEM containing 10% FBS/4 mM L-glutamine (Gibco, GrandIsland, NY, USA)/25 mM HEPES (pH 7.4)/0.5 mM IBMX/10 mM isoproterenol(Tocris bioscience, Ellisville, MO, USA) at 37 C. When lipolytic activity wasanalyzed, 2% fatty acid-free BSA was used instead of 10% FBS.

To inhibit FA re-esterification, differentiated 3T3-L1 adipocytes were pre-incubated with growth medium (DMEM/10% FBS supplemented with 5 mg/mlinsulin) containing 1.2 mM 2-bromooctanoate (TCI, Tokyo, Japan) and 10 mMtriacsin C (Biomol, Plymouth Meeting, PA, USA) for 1 hr. Lipolytic stimulationwas applied with DMEM containing 2% fatty acid-free BSA, 20 mM HEPES(pH 7.4), 0.5 mM IBMX, 10 mM isoproterenol, 1.2 mM 2-bromooctanoate, and10 mM triacsin C.

To inhibit ATGL activity, the cells were treated as above, except that 25 mMbromoenol lactone (BEL) (Cayman, Ann Arbor, MI, USA) was added to themedium instead of 2-bromoactanoate and Triacsin C.

Time-lapse imaging of LD-associated proteins

3T3-L1 adipocytes stably expressing GFP-fused perilipin, CGI-58, GFP-HSL, andGFP-ATGL was differentiated on 35 mm glass-bottom culture dishes. Time-lapseimages during lipolysis were obtained through an oil-immersion objective lens(UApo 40/NA1.35, Olympus, Tokyo, Japan) in the DeltaVision microscope systemplaced in a temperature-controlled room (37 C), as described previously(Haraguchi et al., 1999).

CARS microscopy

3T3-L1 adipocytes were differentiated on 35 mm glass-bottom culture dishes, and200 mM deuterium-labeled palmitate ([D31]-palmitate) (Isotec, St. Louis, MO,USA) was added to DMEM and allowed to be incorporated into large LDs byincubation for 24 hrs. During stimulation of lipolysis, no exogenous FA was addedin the media. CARS imaging experiments were performed by multiplex CARSmicrospectroscopy, on which the details have been described elsewhere (Okunoet al., 2010). All experiments were performed at room temperature.

Immunofluorescence microscopy

All reagents for microscopy were obtained from Wako (Osaka, Japan) unlessotherwise mentioned. For immunostaining of LD-associated proteins, differentiated3T3-L1 cells were fixed with PBS containing 3.7% paraformaldehyde (PFA) for15 min at room temperature. Fixed cells were permeabilized in 0.2% Triton X-100/PBS for 10 min and blocked with 1% BSA/PBS. In the case of digitonin treatment,cells were treated with PBS containing 0.01% digitonin for 5 min on ice andfollowed by fixation with 3.7% PFA/PBS. Cells were then incubated with primaryantibodies against HSL (Cell Signaling, Danvers, MA, USA), ATGL (CellSignaling, Danvers, MA, USA), perilipin (Progen, Heidelberg, Germany), CGI-58/ABHD-5 (Santa Cruz biotechnology, Santa Cruz, CA, USA), and Myc (MBL,Nagoya, Japan) overnight, washed with PBS, and incubated with Alexa 488-, 546-,633- (Molecular Probes, Grand Island, NY, USA), Cy3- or fluoresceinisothiocyanate (Jackson ImmunoResearch, West Grove, PA, USA)-conjugatedsecondary antibody for 1 hr. After washing with PBS, cells were mounted andobserved under a fluorescence microscope (Biozero, Keyence, Osaka, Japan)(Yamaguchi et al., 2007) or a confocal microscope (FV 1000, Olympus, Tokyo,Japan).

Subcellular fractionation

All reagents for subcellular fractionation were obtained from Wako (Osaka, Japan)unless otherwise mentioned. Subcellular fractionation was performed as describedby Liu et al. (Liu et al., 2004), with some modifications. Differentiated 3T3-L1cells from five 10-cm dishes were washed with PBS and harvested in 2 ml of ice-cold buffer A: 25 mM tricine-HCl, pH 7.6, 250 mM sucrose, 10 mM sodiumfluoride, containing 1 mM PMSF (Sigma-Aldrich, St. Louis, MO, USA), aprotease inhibitor mixture (Sigma-Aldrich, St. Louis, MO, USA), and phosphataseinhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and incubated on ice for20 min. Cells were then homogenized using Potter-Elvehjem tissue homogenizer(AS ONE, Osaka, Japan) on ice with ten gentle strokes with the motor-drivenpestle at 2500 rpm. Postnuclear supernatant (PNS) fraction was obtained bycentrifugation at 1000 g for 5 min. PNS was added to a Hitachi 5PA tube, andoverlaid with 3.5 ml of buffer B (20 mM HEPES-NaOH, pH 7.4, 100 mM KCl,and 2 mM MgCl2), and then centrifuged at 40,000 g for 3 hrs at 4 C (Himac CS100GXII, Hitachi, Tokyo, Japan). Lipid droplet (LD) fraction, concentrated in awhite band at the top of the gradient, and cytosolic (Cyt) fraction were collected,and mixed with cold 100% acetone, and incubated at 230 C overnight. The pellet(Pel) fraction was rinsed with PBS and resuspended in buffer C (10 mM Tris-HCl,

pH 7.4, 1 mM EDTA, 0.1% Triton X-100). These fractions were subjected to

SDS-PAGE.

Immunoprecipitation

All reagents for immunoprecipitation were obtained from Wako (Osaka, Japan)unless otherwise mentioned. Differentiated 3T3-L1 cells containing exogenously

expressed Flag-CGI-58 from five 10-cm dishes with or without 2 hrs lipolyticstimulation were washed with PBS and harvested in 2 ml of ice-cold lysis buffer:

25 mM HEPES, pH 7.4, 150 mM NaCl, 1 mM EDTA, 0.1% digitonin containing1 mM PMSF (Sigma-Aldrich, St. Louis, MO, USA), a protease inhibitor mixture

(Sigma-Aldrich, St. Louis, MO, USA), and phosphatase inhibitor cocktail (Sigma-Aldrich, St. Louis, MO, USA), and incubated on ice for 20 min. Cells were then

gently pipetted and centrifuged at 10000 g for 5 min at 4 C (CF 15RXII, Hitachi,

Tokyo, Japan). Immunoprecipitation of Flag-CGI-58 was performed by usingDynabeads Protein G (Life Technologies, Carlsbad, CA, USA) for the cell lysates

by following manufacturer’s instructions. Precipitants performed with an anti-Flagantibody (Wako, Osaka, Japan) were eluted by Flag peptide (Wako, Osaka, Japan)

at a concentration of 0.5 mg/ml in TBS, and endogenous ATGL in theimmunoprecipitate was analyzed. Normal IgG (Santa Cruz Biotechnology, Santa

Cruz, CA, USA) was used as a nonspecific control.

Western blotting

3T3-L1 cells were washed with PBS and directly dissolved in the heated SDS-

PAGE sample buffer. Aliquots of the extracts were SDS-PAGE and protein wastransferred to a nitrocellulose membrane. The blots were probed with antibodies to

CGI-58 (Santa Cruz or self-made (Yamaguchi et al., 2007)), p-ACC (Ser79) (CellSignaling, Danvers, MA, USA), p-HSL (Ser565) (Cell Signaling, Danvers, MA,

USA), HSL (Cell Signaling, Danvers, MA, USA), p-AMPK (Thr172) (CellSignaling, Danvers, MA, USA), ATGL (Cell Signaling, Danvers, MA, USA),

perilipin (Progen, Heidelberg, Germany), and DGAT1 (abcam, Cambridge, MA,USA). Signals were detected by the ECL method (GE Healthcare Life Science,

Fairfield, CT, USA).

Release of glycerol and FA

3T3-L1 cells were grown in 12-well dishes. Differentiated cells were washed twicewith Hank’s buffer and incubated with DMEM containing 2% fatty acid-free BSA/

20 mM HEPES-NaOH (pH 7.4) with or without 0.5 mM IBMX and 10 mM

isoproterenol at 37 C. After incubation, aliquots of the medium were collected atappropriate times, and assayed for glycerol and FA contents using TG E-test kit

and NEFA C-test kit, respectively (Wako, Osaka, Japan).

Measurements of TAG storage and LD diameter

3T3-L1 cells were grown in 12-well dishes. For TAG measurements, differentiatedcells were washed twice with Hank’s buffer and treated with 0.5 mM IBMX and

10 mM isoproterenol for 6 hrs with or without 2-bromooctanoate and triacsin C,

washed with PBS and harvested in a lysis buffer (50 mM Tris-HCl, pH 7.4,150 mM NaCl, 2 mM EDTA, 1% Triton X-100). TAG was measured using a TG

E-test kit (Wako, Osaka, Japan). For LD diameter measurements, differentiatedcells were washed twice with Hank’s buffer and treated with 0.5 mM IBMX and

10 mM isoproterenol for 6 hrs with or without 2-bromooctanoate and triacsin C,washed with PBS and fixed with PBS containing 3.7% PFA for 15 min at room

temperature. LD diameters were measured for phase-contrast microscopic imagesunder each condition. The diameters of the three largest LDs in at least 100 each

adipocytes (more than 300 LDs in total) under each condition were measured(Miyoshi et al., 2008).

Statistical analysis

Statistical analysis was performed by unpaired t-tests or using either one- or two-

way analysis of variance, as appropriate. Bonferroni/Dunn post-hoc test was used

in the event of a significant (P,0.05) ratio. All results are presented as means 6

s.e.m.

AcknowledgementsWe are very grateful to Dr G. C. Henderson for critical reading ofmanuscript. We also thank Dr L. Philippe and Dr V. Couderc forproviding the laser source.

FundingThis work was supported in part by a Grant-in-Aid for ScientificResearch [grant number 19370056 to T.O.]; a Grant-in-Aid forYoung Scientists (B) [grant number 22700652 to T.H.] from theJapan Society for Promotion of Science; and by the Global Center ofExcellence Program (Picobiology, Life Science at the Atomic

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Level), Ministry of Education, Culture, Sports, Science, andTechnology, Japan.

Supplementary material available online at

http://jcs.biologists.org/lookup/suppl/doi:10.1242/jcs.113084/-/DC1

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