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J. Biol. Chem., Vol. 280, Issue 24, 22624-22631, June 17, 2005

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Activation of the Lipid Droplet Controls the Rate of Lipolysis of Triglycerides in the Insect Fat Body^*

Rajesh T. Patel, Jose L. Soulages, Balaji Hariharasundaram, and Estela L. Arrese

From the Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, Oklahoma 74078

Received for publication, November 22, 2004 , and in revised form, March 28, 2005.

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
The hydrolysis of triglyceride (TG) stored in the lipid droplets of the insect fat body is under hormonal regulation by the adipokinetic hormone (AKH), which triggers a rapid activation cAMP-dependent kinase cascade (protein kinase A (PKA)). The role of phosphorylation on two components of the lipolytic process, the TG-lipase and the lipid droplet, was investigated in fat body adipocytes. The activity of purified TG-lipase determined using in vivo TG-radiolabeled lipid droplets was unaffected by the phosphorylation of the lipase. However, the activity of purified lipase was 2.4-fold higher against lipid droplets isolated from hormone-stimulated fat bodies than against lipid droplets isolated from unstimulated tissue. In vivo stimulation of lipolysis promotes a rapid phosphorylation of a lipid droplet protein with an apparent mass of 42–44 kDa. This protein was identified as "Lipid Storage Droplet Protein 1" (Lsdp1). In vivo phosphorylation of this protein reached a peak 10 min after the injection of AKH. Supporting a role of Lsdp1 in lipolysis, maximum TG-lipase activity was also observed with lipid droplets isolated 10 min after hormonal stimulation. The activation of lipolysis was reconstituted in vitro using purified insect PKA and TG-lipase and lipid droplets. In vitro phosphorylation of lipid droplets catalyzed by PKA enhanced the phosphorylation of Lsdp1 and the lipolytic rate of the lipase, demonstrating a prominent role PKA and protein phosphorylation on the activation of the lipid droplets. AKH-induced changes in the properties of the substrate do not promote a tight association of the lipase with the lipid droplets. It is concluded that the lipolysis in fat body adipocytes is controlled by the activation of the lipid droplet. This activation is achieved by PKA-mediated phosphorylation of the lipid droplet. Lsdp1 is the main target of PKA, suggesting that this protein is a major player in the activation of lipolysis in insects.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Insects rely on lipid reserves to survive during physiological non-feeding periods or to meet the energy requirements of developing eggs, flight, and starvation. The fat body is the principal site for storage of both glycogen and lipids. The fat body synthesizes most of the proteins found in the hemolymph while also serving as the main storage site of triglycerides (TG),¹ which constitute >90% fat body lipids. Therefore, functionally, the fat body accomplishes roles that in vertebrates are carried out by both liver and adipose tissue (1).

The tobacco hornworm, Manduca sexta, feeds constantly, and the content of fat body TG increases continuously until the end of the larval development. During the larval period (20 days), the content of TG in the fat body increases from a few micrograms to 80 mg (2). During subsequent development, the lipid reserves are used to sustain the life of the adult insect, which feeds occasionally (2–5). Due to these metabolic features, M. sexta represents an excellent model for studying the basic mechanisms involved in either the synthesis/deposition of TG in larvae or the mobilization of TG in adult insects (moth).

TG is stored in fat body adipocytes as cytosolic lipid droplets (6). TG hydrolysis (lipolysis) is mediated by a TG-lipase that has been purified from the cytosol (7). Like hormone-sensitive lipase (HSL), which catalyzes the rate-limiting step in mobilization of adipose tissue fatty acids (8, 9), the TG-lipase from M. sexta fat body is an enzyme that can be phosphorylated. The end product of insect lipolysis is sn-1,2-diacylglycerol (DG) that is released into the hemolymph (10, 11) and loaded into the hemolymph lipoprotein, lipophorin. This causes the transformation of high density lipophorin (HDLp) into low density lipophorin (LDLp), which transports DG to the sites of utilization, e.g. the flight muscle, and ovaries, where it is hydrolyzed to fatty acids by a lipophorin-lipase (12).

The lipolytic process is under hormonal regulation by the neuropeptide adipokinetic hormone (AKH) (13). AKH action is comparable to that of glucagon in mammals. It contributes to hemolymph sugar homeostasis and is also involved in the mobilization of sugar and lipids from the fat body during energy-requiring activities (14–17). AKH receptors from the fruitfly Drosophila melanogaster and the silkworm Bombyx mori have been recently identified (18). These receptors are related to the mammalian gonadotropin-releasing hormone receptor, which is a G protein-coupled receptor that activates both inositol phosphate and cAMP signaling responses. In M. sexta, AKH mobilizes glycogen during the larval stages and promotes a massive lipolytic response in the adult stage (19). AKH promotes a rapid activation of fat body cAMP-dependent protein kinase A (PKA). Supporting the role of PKA in lipolysis, agents that raise intracellular cAMP concentration, such as 8-Br-cAMP and forskolin, also stimulate lipolysis in the adult insect (20). Besides the involvement of cAMP, the lipolytic response of AKH also induces a sustained increase in calcium influx (20–22). Moreover, calcium-mobilizing agents such as thapsigargin or ionomycin strongly stimulate lipolysis (20). Therefore, unlike the vertebrate system in which the lipolytic process is activated by cAMP (23) and inhibited by intracellular calcium (24), in insects, both messengers (cAMP and calcium) stimulate lipolysis. Protein phosphorylation mediated by PKA is expected to be part of the mechanism controlling the activation of lipolysis, but the nature of the proteins targeted by PKA remains to be elucidated. It has been shown that TG-lipase can be phosphorylated in vitro by purified PKA from the insect fat body. However, this phosphorylation failed to increase the enzyme activity when assayed in vitro with an artificial substrate (25). In the present study, the mechanism of activation of lipolysis was investigated using the native substrate of the TG-lipase, the lipid droplet, and a combination of in vivo and in vitro experiments. To study the role of PKA and the impact of the phosphorylation of the TG-lipase and the lipid droplet proteins on the activation of lipolysis, we used a novel approach in which the lipolytic process was reconstituted in vitro using in vivo TG-radiolabeled lipid droplets as the substrate of purified TG-lipase and purified insect PKA. The studies presented here confirm the presumed role of PKA in activation of the lipolysis. More interestingly, the studies suggest that, contrary to previous assumptions, the main role of PKA is to activate the substrate of lipolysis, the lipid droplet, rather than the TG-lipase. The studies led to the identification of the Lsdp1 as the main target of PKA activation. Moreover, the phosphorylation of Lsdp1 emerged as a likely regulator of the lipolytic activation.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Materials—[³²PO₄]Orthophosphate and [-³²PO₄]ATP were purchased from MP Biochemicals (Irvine, CA). Labeled trioleoylglycerol ([tri-9,10-³H(N)]oleoylglycerol) and [9,10-(n)³H]palmitic acid were from PerkinElmer Life Sciences. Protease and phosphatase inhibitors were purchased from Sigma. DEAE-Sepharose Fast Flow, phenyl-Sepharose, and Q-Sepharose were from Amersham Biosciences. Hydroxyapatite Bio-Gel HT Gel was purchased from Bio-Rad. M. sexta AKH was obtained from Peninsula Laboratories (Belmont, CA). Electrophoresis items were from Invitrogen. Silica Gel G plates were purchased from J. T. Baker (Phillipsburg, NJ). Trypsin sequencing grade was purchased from Promega (Madison, WI). All of the other chemicals were of analytical grade.

Experimental Insects—M. sexta eggs were purchased from Carolina Biological supplies, and larvae were reared on artificial diet (26). Adult insects were maintained at room temperature without food. All of the experiments were carried out using adult insects 48–72 h after emergence. To achieve a consistent basal level of lipolysis, the insects were decapitated 24 h ahead of the experiment. Two hours before the experiments, the insects were injected with 13 mg of trehalose dissolved in 20 µl of water (27).

Purification of TG-lipase—TG-lipase was purified from the cytosolic fraction of M. sexta fat body homogenates using anion-exchange, hydroxyl-apatite, and hydrophobic interaction chromatography as reported previously (7).

Purification of the Catalytic Subunit of cAMP-dependent PKA—The catalytic subunit of PKA was purified from the cytosolic fraction of M. sexta fat body homogenates using a combination of ion-exchange chromatographies, as previously described (25).

Subcellular Fractionation—Fat body tissue from two insects was combined and homogenized with a Potter-Elvehjem glass homogenizer fitted with Teflon pestle using 6 ml of homogenization buffer (20 mM Tris, pH 7.4, 0.25 M sucrose, 1 mM EDTA, 0.1 mM benzamidine, 10 mg/liter leupeptin, 1 mg/liter aprotonin, 0.1% 2-mercaptoethanol, 2 mM imidazole, 2 mM sodium fluoride, 1.5 mM sodium molybdate, 1 mM sodium orthovanadate, and 4 mM sodium potassium tartrate). The homogenate was overlaid with 2 ml of buffer without sucrose and centrifuged (100,000 x g for 1 h). Three fractions were collected: fat cake; infranatant; and pellet. To ensure that the lipid droplets were free of cytosolic components, the fat cake was resuspended in homogenization buffer and gently vortexed. The sucrose concentration was adjusted to 15% (w/v), and a layer of 2-ml buffer without sucrose was laid on top. Samples were centrifuged in a SW 40 rotor at 100,000 x g for 1 h. Purified lipid droplets were collected from the top and resuspended in homogenization buffer without sucrose. Typically, lipid droplets of two insect fat bodies were resuspended in 0.5 ml of buffer. The pellet was resuspended in buffer and recentrifuged at 100,000 x g for 1 h. The resulting pellet was dissolved in 1 ml of buffer and centrifuged at 500 x g for 15 min. The resulting supernatant was used as membranes fraction. The infranatant (cytosolic fraction) was passed through a small Q-Sepharose column equilibrated with buffer (10 mM Na₂HPO₄, pH 7.4, 1 mM EDTA, 0.1% (v/v) 2-mercaptoethanol, 0.1 mM benzamidine, 0.37 mM Triton-X-100, 10 mg/liter leupeptin, 1 mg/liter aprotonin, 2 mM imidazole, 2 mM sodium fluoride, 1.5 mM sodium molybdate, 1 mM sodium orthovanadate, and 4 mM sodium potassium tartrate). The column was extensively washed with equilibration buffer, and the proteins were eluted with a NaCl gradient (20–150 mM) in the same buffer. TG-lipase was eluted with 180 mM NaCl in the same buffer, and all of the fractions containing TG-lipase activity were pooled, dialyzed, and concentrated to a final volume of 4 ml.

In Vivo Protein Phosphorylation Studies—Experimental insects were injected with 250 µCi of [³²PO₄]orthophosphate (carrier free) and 90 min later with 100 pmol of AKH. Fat body tissue was dissected at various times after the hormonal injection. For each time, tissue from two insects was pooled and homogenized. The lipid droplets were isolated as indicated above. The lipid droplet-associated proteins were separated by SDS-PAGE on 10% gels according to Laemmli but in sample buffer containing 6% (w/v) SDS. Proteins were stained with Coomassie Blue. The gel was dried, and phosphorylation was visualized by autoradiography. Gels and autoradiograms were scanned on an imaging densitometer (Bio-Rad model GS-700). The intensity of protein bands and phosphorylation was quantified from the gel and autoradiogram scans, respectively, using the Multi Analyst Macintosh software.

Protein Identification by Mass Spectrometry—³²P-Phosphorylated samples of lipid droplets were separated by two-dimensional electrophoresis according to the method of O'Farrel. The pH gradient ranged between 4.0 and 9.0, and the second dimension was done on 10% acrylamide gels. Coomassie Blue-stained gels were dried and exposed. The highly phosphorylated spot with an apparent mass of 42 kDa was excised from the gel. The protein was cleaved with trypsin and then sequenced by microcapillary reverse-phase high pressure liquid chromatography nanoelectrospray tandem mass spectrometry on a Finnigan LCQ DECA XP Plus quadrupole ion trap mass spectrometer at the Harvard Microchemistry Facility. The MS/MS spectra were correlated with known sequences using the algorithm Sequest and programs were developed in that laboratory (28). For peptide mass fingerprinting of 42 and 44 kDa, lipid droplet-associated proteins were separated on 10% SDS-PAGE and the bands visualized by Coomassie Blue staining, excised, minced, and destained using 100% acetonitrile followed by four washes with water. The gel pieces were incubated for 20 min in 500 µl of 100 mM ammonium bicarbonate followed by 20 min incubation with 500 µl of 50% acetonitrile in 50 mM ammonium bicarbonate. Gel pieces were dried under vacuum, rehydrated, and digested with 50 ng/µl trypsin in 25 mM ammonium bicarbonate at 4 °C overnight. Peptides were extracted and analyzed by MALDI-TOF mass spectrometry in the Core Facility using the department ABI Voyager De PRO MALDI-MS and -cyano-4-hydroxycinnamic acid as matrix.

Preparation of Endogenously [³³H]TG-labeled Lipid Droplets—Fat body lipids were radiolabeled following a long-term procedure previously reported (11, 29). During the fifth larval instar, insects were fed 200 µCi of [9,10(n)-³H]palmitic acid and, after completion of development (32 days), adult insects were decapitated and injected with trehalose as indicated above. Lipolysis was stimulated by injection of 100 pmol of AKH, whereas injection of buffer provided the level of basal lipolysis. Fat bodies were dissected 20 min after injection, and the lipid droplets were isolated as described above. Lipid analysis of TG showed that the vast majority of the radiolabel (99.8%) was localized in the fatty acyl residues. The remaining 0.2% was found in the glycerol backbone. sn-1,3 positions of TG contained 88.4 ± 2% of the label. An analysis of the distribution of radioactivity among different neutral lipid classes of the lipid droplets showed that TG contained 97.4 ± 0.5% radioactivity.

TG-Lipase Activity against [³H]TG-Lipid Droplets—An aliquot of the lipid droplet preparation containing 100 nmol of TG was transferred to a glass tube containing lipase reaction buffer. The reaction was initiated by adding purified TG-lipase (7 µg) in a final volume of 150 µl. Final reaction conditions were 50 mM Tris, pH 7.9, 500 mM NaCl, 0.02% (w/v) bovine serum albumin, 2 mM dithiothreitol, 0.67 mM TG, and 0.37 mM Triton X-100. The mixture was gently vortexed for 20 s and incubated at 37 °C with constant shaking. After 30 min, the reaction was terminated by the addition of 750 µl of chloroform:methanol (2:1) and 5 µl of 6 N HCl. The mixture was vortexed for 1 min and centrifuged at 2000 x g for 2 min. The organic phase was collected, and counts/min were calculated in an aliquot. The remaining organic phase was dried under a stream of nitrogen, and the lipids were separated by thin-layer chromatography on Silica Gel G plates using hexane:ethyl ether:formic acid (70:30:3) as the developing solvent (29). The monoglyceride, DG, free fatty acid, and TG fractions were visualized by I₂ vapors and scraped from the plates. After complete removal of the I₂, the radioactivity associated with each fraction was determined by liquid scintillation counting. Blank reactions in which the TG-lipase was omitted were used to obtain the basal level of distribution of radioactivity among the lipid classes and calculate the percentage of hydrolysis. The lipase activity was expressed as nmol of TG hydrolyzed/min mg protein. All of the determinations were carried out in duplicate. Aliquots of lipid droplets resuspended in TG-lipase reaction buffer were freshly stained with Oil Red O and were observed under the microscope. We found numerous red-colored spheres (0.6–3.5 µm), confirming the existence of the lipid droplets in the reaction mixture. The size of lipid droplets in the final preparation was slightly smaller than the size of native lipid droplets (1.2–4.1 µm).

In Vitro Phosphorylation Reactions of TG-Lipase and Lipid Droplets—The lipid droplets were phosphorylated in a reaction mixture (90 µl) containing kinase reaction buffer (50 mM MOPS, 1 mM magnesium acetate, 0.5 mM EDTA, and 2 mM dithiothreitol), purified PKA (0.25 units), 7 µl of lipid droplets (0.1 µmol TG), and 0.2 mM [-³²PO₄]ATP (5 x 10⁶ cpm/nmol). After 20 min of incubation at room temperature, the reaction was terminated by the addition of electrophoresis sample buffer and analyzed by SDS-PAGE on 4–20% acrylamide gels followed by autoradiography. ³²P-Labeled protein band profile was performed by densitometric analysis.

Purified TG-lipase was phosphorylated by insect PKA in the presence of [-³²PO₄]ATP as described above. Phosphorylation of the lipase was confirmed by SDS-PAGE and autoradiography.

To study the effect of phosphorylation on the TG-lipase activity, [³H]TG-lipid droplets and/or TG-lipase were phosphorylated as described above but using unlabeled ATP. The reactions were terminated by the addition of 5 mM EDTA.

Measurement of Low Density Lipoprotein/High Density Lipoprotein Ratio of Hemolymph—The content of DG in the hemolymph was assessed by measuring the relative mass of LDLp and HDLp after separation of the lipoproteins by ultracentrifugation in a KBr density gradient (27). The LDLp/HDLp ratio was used as an index of lipolysis.

Western Blotting—Polyclonal antibodies against purified TG-lipase were raised in chicken at Cocalico Biologicals (Reamstown, PA). For Western blotting, proteins were separated by SDS-PAGE (10%) and transferred to nitrocellulose membranes. Immunodetection was performed using anti-TG-lipase antibodies (1:200). After incubation of membranes with horseradish peroxidase-conjugated rabbit anti-chicken secondary antibody (1:50,000), peroxidase activity was detected using ECL chemiluminescence reagents (Amersham Biosciences). X-ray films were scanned, and the intensity of the positive signal was quantified by densitometry.

Protein and TG Content of Lipid Droplets—The lipid droplet-associated proteins were precipitated with acetone (85% v/v) at –20 °C for at least 2 h. Afterward, samples were centrifuged at 10,000 x g for 5 min and the resultant pellet was dissolved in final 10% SDS (w/v). An aliquot was used to measure total protein using the BCA method (Pierce).

TG was determined using the Infinity triglyceride reagent kit as described by the manufacturer (ThermoTrace Ltd, Melbourne, Australia). Triolein was used as standard for the calibration curve.

Statistics—Statistical comparisons were made by the Student's t test. p < 0.05 was considered to be significant.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Study of a Potential Translocation of the Lipase from the Cytosol to the Lipid Droplet—Studies carried out in rat and 3T3 L1 adipocytes have shown that activation of lipolysis triggers the translocation of HSL from the cytosol to the lipid droplets (30, 31). The potential role of this mechanism of lipolysis activation in M. sexta fat body was investigated by determining the relative distribution of TG-lipase between the cytosolic and lipid droplet fractions of the adipocytes. Immunoblotting was used to estimate the abundance of TG-lipase in the fractions. A comparison between the fractions obtained from control insects and insects treated with AKH for 20 min indicated that activation of lipolysis does not change the levels of cytosolic TG lipase (Fig. 1). Moreover, we were not able to detect the presence of TG-lipase in the lipid droplet fractions. Regardless of the lipolytic condition, the fat body TG-lipase was exclusively found in the cytosol and its abundance was unaffected by the level of lipolysis (Fig. 1).

View larger version (30K): [in this window] [in a new window]   FIG. 1. Subcellular distribution of TG-lipase among cytosol, lipid droplet, and membranes fractions under basal and stimulated lipolysis. Cytosol, membranes, and lipid droplets from the fat body of control insects (C) and AKH-stimulated insects (AKH) were separated on 8% SDS-PAGE and analyzed by Western blotting using anti-serum against TG-lipase. Normalization among control and stimulated samples for each subcellular fraction was accomplished loading equal percentages of each fraction onto each lane (0.5% total for cytosol, 2% total for lipid droplets, and 0.4% for membranes).

Phosphorylation of the lipase did not modify the enzyme activity hydrolyzing TG contained in the lipid droplets from control insects (Fig. 2). However, the activity of the lipase against lipid droplets isolated from AKH-treated insects was significantly greater (2.4-fold increase) than that measured against lipid droplets obtained from control insects (Fig. 2).

It must be noted that no significant endogenous lipase activity was detected in lipid droplets from control- or AKH-treated insects. This was determined by incubating the lipid droplets in the reaction buffer but in the absence of TG-lipase for different time periods (0–60 min). This result is consistent with the absence of lipase associated to the lipid droplets inferred from the Western blotting analysis (Fig. 1).

Time Course of AKH-induced Activation and Phosphorylation of the Lipid Droplets—AKH-induced activation of lipolysis involves a rapid 4-fold increase of fat body PKA activity that is reached 2–5 min after the injection of the hormone into the hemolymph (20). Potential changes in the phosphorylation state of the proteins of the lipid droplets and its possible correlation with the "activity" of the lipid droplets against purified TG-lipase were investigated. To study the time course of changes in protein phosphorylation together with the lipase activity measured against TG contained in the lipid droplets, we used lipid droplets containing both radiolabeled TG and ³²P-radiolabeled proteins isolated from insects fed with [³H]palmitic acid and injected with [³²P]orthophosphate 90 min prior to the experiments.

As shown in the Fig. 3, the activity of purified TG-lipase was dependent on the time of isolation of the lipid droplets. Rapid and significant changes in the sensitivity of the lipid droplets toward the lipase were observed as early as 5 min after the hormonal stimulation. The highest lipase activity (2.6-fold increase over basal) was observed against the lipid droplets that were isolated 10 min after the stimulation of lipolysis.

³H/³²P-radiolabeled lipid droplets were also used to examine the time-dependent changes in the pattern of [³²P]protein phosphorylation. Fig. 4A depicts the protein profiles of lipid droplets separated in 10% SDS-PAGE. The autoradiogram of the same SDS-PAGE gel is shown in the panel B. Five minutes after the stimulation of lipolysis, the only significant change in phosphorylation was observed in a band of 43 kDa (2.7-fold increase). The change in phosphorylation of the 43-kDa band is very rapid, reaching a maximum of 10 min after the injection of the hormone (Fig. 5, A–C). Densitometric analysis of the gel indicated that the 43-kDa band of the autoradiogram corresponded to two close protein bands migrating with apparent masses of 42.8 and 44.2 kDa (Fig. 4B).

View larger version (13K): [in this window] [in a new window]   FIG. 2. Effect of TG-lipase phosphorylation and AKH induced changes in the substrate on the lipase activity, hydrolyzing TG present in the lipid droplets (LD). Purified TG-lipase from fat body tissue was in vitro phosphorylated by incubation with fat body cAMP-dependent PKA. The activity of purified TG-lipase was measured against in vivo radiolabeled ([3H]TG)-lipid droplets isolated from insects with basal lipolysis (LD Control). The effect of AKH on the substrate properties was studied using lipid droplets isolated from insects with stimulated lipolysis (20 min after injection of AKH) (LD AKH). Enzyme activities are expressed in nmol TG hydrolyzed/min mg protein. Data represent the mean ± S.E. (n = 4). *, p < 0.05 versus control lipid droplets.

View larger version (22K): [in this window] [in a new window]   FIG. 3. Time course of the activation of the substrate induced by AKH on the activity of TG-lipase. [3H]TG lipid droplets were isolated from insects with basal lipolytic activity (0) and AKH-stimulated insects (5, 10, 20, and 30 min after treatment). Aliquots containing 100 nmol of TG were used to measure the TG-lipase activity. Incubations were done for 30 min at 37 °C as indicated under "Experimental Procedures." Data are expressed in nmol TG hydrolyzed/min mg protein and represent the mean ± S.E. (n = 4). *, p < 0.05 versus control.

Changes in the phosphorylation of other protein bands with apparent masses of 52, 66, 95, and 110 kDa were also observed. However, no correlation was found with the phosphorylation of these proteins and the lipase activity. For instance, phosphorylation of the 52-, 66-, and 110-kDa bands became more relevant at 20 and 30 min.

As expected, the changes in lipase sensitivity of the lipid droplets preceded the lipolytic response measured by the increase in the content hemolymph DG that was estimated as the ratio of hemolymph LDLp and HDLp, respectively (Fig. 5D). In these insects, the onset of lipid mobilization from the fat body into the hemolymph begins 10 min after the injection of 100 pmol of AKH (27).

View larger version (71K): [in this window] [in a new window]   FIG. 4. Time-dependent changes in phosphoproteins of the lipid droplets. [32P]Lipid droplets were isolated at different times after hormonal treatment and subjected to SDS-PAGE in 10% acrylamide gels. A, Coomassie Blue-stained gel. B, autoradiogram of the gel shown in A. Control lipid droplets were loaded in lanes labeled 0. The remaining lanes were loaded with lipid droplets isolated 5, 10, 20, and 30 min after the injection of AKH, as indicated in the figure. Approximately 15 µg of protein was loaded into each lane. The intensity of protein bands and phosphorylation was quantified from the gel and autoradiogram scans, respectively, using an imaging densitometer as explained under "Experimental Procedures." The phosphorylation state was calculated as the ratio between the intensity of phosphorylation and the intensity of the protein band.

View larger version (32K): [in this window] [in a new window]   FIG. 5. Correlation between the phosphorylation of the lipid droplets and their activity as TG-lipase substrates. Panels A–C show the densitometric scans of the Coomassie Blue-stained gel (shown in Fig. 4A) and corresponding autoradiogram (shown in Fig. 4B) for 0 (control), 5, and 10 min, respectively. The scans of the autoradiography (AR) are represented with solid lines. The scans of the gel are shown with broken lines. The major phosphoprotein has an apparent mass of 42 kDa (52 mm). Other phosphoproteins are as follows: 52 kDa (43 mm); 66kDa (36 mm); 95kDa (28 mm); and 110kda (26 mm). Panel D, the time course of the AKH-induced changes in substrate activity showed in Fig. 4 is correlated with the time-dependent changes in the phosphorylation state of the 42-kDa lipid droplet-associated protein. The changes in phosphorylation state and lipase activity were expressed as fold increase over the basal condition. The ratio of LDLp to HDLp is proportional to the level of circulating DG. The increase in the LDLp/HDLp ratio is the consequence of AKH-induced lipolysis. Data are the mean ± S.E.; n = 4 for lipase activity, n = 3 for phosphorylation of 42-kDa protein and LDLp/HDLp ratio.

In Vitro Activation of Lipolysis from Purified TG-Lipase, PKA, and Lipid Droplets—The phosphorylation profiles of the lipid droplet-associated proteins obtained upon incubation of lipid droplets isolated from control insects (basal lipolysis) with purified fat body PKA in the presence of [³²P ]ATP were compared with the phosphorylation profiles observed in vivo (Fig. 6). This comparison showed similarities between the pattern of proteins phosphorylated in vitro by PKA and the pattern of phosphorylation observed in vivo when the lipolysis was stimulated by AKH. As observed in in vivo experiments, Lsdp1 was also the major phosphoprotein of the lipid droplet after in vitro phosphorylation, indicating that Lsdp1 is the main substrate of PKA in the lipid droplets.

To study the role of PKA on the activation of lipolysis, [³H-TG]lipid droplets (from insects with basal lipolysis) were incubated with or without purified PKA and ATP. Following phosphorylation, the lipid droplets were incubated with purified lipase and the enzyme activity was determined. The results showed that in vitro phosphorylation of the lipid droplets with PKA promotes a significant enhancement (2-fold) of the lipase activity (Fig. 7).

View larger version (20K): [in this window] [in a new window]   FIG. 6. Comparison between the patterns of protein phosphorylation of the lipid droplets observed in vivo (AKH-induced) and in vitro (PKA-catalyzed phosphorylation). Densitometric profiles of the autoradiograms of [32P]phosphoproteins of the lipid droplets. In vivo phosphorylation was induced by AKH injection into insects previously injected with 250 µCi of [32P]orthophosphate. Lipid droplets were isolated after 20 min of AKH treatment. For in vitro phosphorylation, lipid droplets were isolated from control insects (basal lipolysis) and incubated with purified cAMP-dependent PKA and 0.2 mM [-32PO4]ATP (5 x 106 cpm/nmol) for 20 min. Both samples were subjected to SDS-PAGE in 4–20% acrylamide, which provides a good resolution in a broad range of size (6–200 kDa). Autoradiograms were scanned on an imaging densitometer as explained under "Experimental Procedures." The major peak corresponds to the 42-kDa band.

View larger version (12K): [in this window] [in a new window]   FIG. 7. Effect of cAMP-dependent PKA-mediated phosphorylation of lipid droplet (LD) proteins on the activity of TG-lipase and phosphorylated TG-lipase. In vivo radiolabeled ([3H]TG) lipid droplets were isolated from insects with basal lipolytic activity and incubated 20 min with 0.2 mM ATP in the presence of purified PKA (Phosphorylated-LD) or in the absence of PKA (LD). The reaction was terminated by the addition of 5 mM EDTA (final concentration). Subsequently, the samples were used as the substrate for both TG-lipase and phosphorylated TG-lipase. Purified TG-lipase was phosphorylated in vitro by incubation with purified PKA and ATP. Data that are expressed in nmol TG hydrolyzed/min mg protein represent the mean ± S.E. (n = 4). *, p < 0.05 versus LD.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

 
Understanding the regulation of the lipolytic process is essential to the full understanding of the metabolism of triglycerides. The mechanism of basal lipolysis and the mechanism of activation of lipolysis are complex processes whose details are far from being fully understood in any system (32, 33). The complexity of the lipolytic activation is given by the fact that the lipolytic process involves the interaction of an enzyme, whose properties are not well established, with a lipid surface whose structure is not well understood either. The lipid droplet is recognized as an important organelle that accomplishes essential functions in adipocytes and therefore in the homeostasis of lipid metabolism of the organism. The lipid droplets contain a large number of proteins embedded in the lipid surface (34, 35). Many of these proteins could play an active role in the lipolytic response and other metabolic reactions. The importance of studying these proteins is currently recognized. However, none of the potentially important proteins has been fully characterized yet from a functional or structural point of view. Most of the current understanding of the lipolytic process has been achieved from studies carried out in 3T3 adipocytes (32, 33). The current knowledge of lipolysis in insects is far more modest than that achieved in mammalian cells. The current study provided several advances on the mechanism of lipolysis in insects. Moreover, the experimental conditions for an in vitro system that allows the reconstitution of the lipolytic activation were established in this study. This in vitro system combined with the simplicity of carrying out in vivo studies represents an important advance that will facilitate future studies on the molecular mechanism underlying the mobilization of TG stores in insects.

Lipid Droplet Activation Plays a Major Role in the Activation of Lipolysis—Our puzzling inability to observe phosphorylation-dependent activation of the lipase using an artificial substrate (25) led us to study the role of phosphorylation of the lipase using the native substrate, the lipid droplets. We thought that the artificial substrate, an emulsion of TG and Triton X-100 (1:5, mol/mol), prevented the observation of the activation of the lipase upon its phosphorylation. The possibility to obtain homogenously TG-radiolabeled lipid droplets allowed the study of the role of lipase phosphorylation under experimental conditions that resembled the physiological conditions. Even under these conditions, phosphorylation of the lipase did not promote an increase in the lipolytic rate. However, the use of the native substrate led us to the main finding of this work that hormonal stimulation of TG lipolysis in insects involves the activation of the lipolytic substrate, the lipid droplets. The fact that the activity of purified lipase was greater against lipid droplets isolated from insects with high levels of lipolytic activity than against lipid droplets isolated from insects in a basal lipolytic state (Figs. 2 and 3) is an indication that the activation of the substrate could represent the major point of control of the lipolytic rate. This information provides new evidence to the increasing notion that the lipid droplets, as organelles, are active participants in the process of lipolysis.

Phosphorylation of the Lipid Droplet Activates Lipolysis—As shown in Fig. 4, hormonal activation of lipolysis alters the pattern of phosphorylation of the lipid droplets. The changes in phosphorylation are very fast and have a time course that correlates to a good extent with the activation state of the lipid droplets (Fig. 5). These observations suggested a relevant role of the phosphorylation of the lipid droplets in the activation of lipolysis. This was clearly demonstrated when the activation of the lipid droplets was achieved in the reconstituted system containing the purified enzymes, PKA and lipase, and control lipid droplets (Fig. 7). The in vitro assay allowed discarding the potential influence on the rate of lipolysis of other changes in structure and composition of the lipid droplets that could take place in vivo. Moreover, it proved that the combination of TG-lipase, PKA, and lipid droplets without additional cellular machinery reproduced to a great extent the activation observed with lipid droplets obtained from insects treated with AKH.

Inspection of the pattern of phosphorylation of the proteins of the lipid droplets showed that a 42–44-kDa protein was the main target of the phosphorylation cascade triggered by AKH. On the basis of its sequence identity with a full-length sequence from D. melanogaster, this protein was identified as Lsdp1. Lsdp1 is not highly abundant in the lipid droplets of adult insects; however, it is the main phosphoprotein. Lsdp1 was the prevalent target of in vivo phosphorylation, induced by AKH, as well as the main target of PKA when the lipid droplets were phosphorylated in vitro. Moreover, because the changes in phosphorylation of Lsdp1 correlated with the activity of the lipid droplets measured with purified lipase, the present studies suggest that Lsdp1 could play a major role in the activation of TG lipolysis in the insect fat body. This study did not provide information on the role of the phosphorylation of the other phosphoproteins observed in the lipid droplets.

D. melanogaster Lsdp1 has been previously recognized as a protein that shares similarity with the mammalian proteins of the PAT family, which is formed by a group of proteins such as Perilipins (Peri A), ADRP (adipose differentiation-related protein), and TIP47 (tail-interacting 47-kDa protein), that are associated with the lipid droplets (36). Only two insect proteins appear to belong to the PAT family, Lsdp1 and Lsd2 (36, 37).

The M. sexta Lsdp1 sequences were not found in either Drosophila or Anopheles Lsd2 proteins. An analysis of the amino acid sequence of Drosophila Lsdp1 and Lsd2 shows that these two proteins have 31.9% identity in a region of 119 amino acids localized toward the N terminus (residues 11–130 for Lsdp1 and residues 35–154 for Lsd2, respectively). This is the region in which the PAT domain localizes (36, 37). A much lower similarity is found beyond this region. Likewise A. gambiae Lsdp1 and Lsd2 proteins also have a region toward the N terminus with some identity.

Lsdp1 shares a small structural motif, "PAT domain," with the lipid droplet-specific proteins, perilipins, ADRP, and TIP47 (38). The PAT domain is an N-terminal sequence of 100 residues common to a small group of proteins exclusively found in the lipid droplet. Using a restricted perilipin PAT domain shortened to 25 amino acids, Lu et al. (38) identified two insect proteins, Lsdp1 and Lsd2, containing the shortened PAT domain (38). The comparison of the two Drosophila PAT domain-encoding genes, Lsdp1 and Lsd2, with genes present in A. gambiae, B. mori, and Dictyostelium discoideum indicated that insect genomes encode only two PAT domain proteins (37). However, the sequence similarity tree based on amino acid identities showed that insect Lsd proteins cannot be paralleled to any of the vertebrate family members (37). The fact that the sequence elements from M. sexta Lsdp1 shown in Table I matched exclusively with insect Lsd proteins corresponds with that previous observation. The function of the insect Lsd proteins is unknown. It has been shown in transgenic Drosophila that Lsdp1 (36) and Lsd2 (36, 37) proteins localize on the surface of lipid droplets within cells of larval fat bodies, confirming that these distantly related PAT proteins possess the structural elements required for targeting to the lipid droplets (36). Strong overexpression of Lsd2 causes an increase of TG storage in Drosophila, whereas Lsd2 mutant flies are lean, suggesting that Lsd2 operates in a perilipin-like manner (37). It is of interest to note in the alignment of Drosophila Lsdp1 and Lsd2 that there is an absolute identity among certain residues in a very periodic manner, and as it is previously reported (37) that there is a high degree of conservation between these two proteins in their N termini. The fact that M. sexta Lsdp1 is a lipid droplet-associated protein is an additional indication that, in insects, Lsds are lipid droplet components.

Among the PAT proteins, there seems to be some functional similarities between perilipin A and Lsdp1. Other than the results of this study, there are no previous reports on the function of Lsdp1.

Functional and Structural Relationship between Lsdp1 and Perilipin A—Perilipin A (62–67 kDa) is a highly abundant phosphoprotein found in the lipid droplets of mammalian adipocytes (39–41). Unphosphorylated Peri A acts as a barrier to lipases (42–44), thereby decreasing basal lipolysis and increasing intracellular TG storage. Peri null mice have very reduced adipose tissue mass (45, 46). Aside from the fact that Peri A and Lsdp1 share a small region of sequence identity near the N terminus, these two proteins are clearly distinguished by their size, sequence, and cellular abundance. As expected, anti-perilipin antibodies did not recognize Lsdp1 from M. sexta (data not shown). Despite these differences, some known functional properties of Peri A are similar to the function of M. sexta Lsdp1. For instance, Peri A is phosphorylated by PKA (39, 47) and, as shown in a recent study (34), overexpression and phosphorylation of Peri A in Chinese hamster ovary cells lead to a significant lipolytic response. Because Chinese hamster ovary cells do not express HSL, the lipolytic response was attributed to the action of cytosolic lipases triggered by the phosphorylation of Peri A. Considering the fact that we observed a strong correlation between the phosphorylation of Lsdp1 and the lipolysis in vivo and in vitro, the role of Peri A appears highly similar to the function of Lsdp1 inferred from this study. This study provides evidence suggesting that biochemical elements of the mechanism of TG mobilization in animals arose very early in evolution and have been conserved.

Role of PKA in the Activation of Lipolysis—Previous studies have shown that AKH promotes an increase in PKA activity, as determined by the activation of glycogen phosphorylase (13). On the other hand, supporting a role for PKA in the activation of lipolysis, other studies have shown that cAMP analogues and adenylate cyclase activators activate lipolysis (20). Although these previous studies suggested a role of PKA in the activation of lipolysis, they did not provide direct experimental evidence linking PKA and lipolysis. The present study provided direct evidence supporting the role of PKA in the activation of lipolysis. It showed that the pattern of in vitro phosphorylation of the lipid droplets with purified PKA is very similar to that observed upon in vivo stimulation of the insects with AKH (Fig. 6) and that the lipolytic activation can be reconstituted in vitro from isolated lipid droplets and purified PKA and lipase (Fig. 7).

In vitro phosphorylation of TG-lipase catalyzed by PKA did not enhance its activity against the native (Fig. 2) or the artificial substrate (25). This is in contrast with the mammalian HSL for which in vitro phosphorylation with PKA induced a 3-fold increase in lipase activity (48, 49).

Role of Lipase Binding to the Lipid Droplet and Kinetics of TG Hydrolysis on the Activation of Lipolysis—Hormonal stimulation of the lipolytic response in mammalian adipocytes induces the phosphorylation and translocation of HSL to the surface of the lipid droplet. This mechanism is the first critical step of lipolysis and explains at least partially the activation of lipolysis in mammalian adipocytes (30, 31, 32, 33). We did not find evidence indicating the presence of the lipase in the lipid droplet of adipocytes obtained from insects with either basal or high lipolytic rates. This result indicates that the activation of lipolysis in insects does not involve a tight association of the TG-lipase with its substrate, the lipid droplets. In other words, translocation of the enzyme to the lipid droplet does not seem to take place. If it does, it involves a low binding affinity association, such that the enzyme would dissociate from the lipid droplet during the preparation of the homogenate and centrifugation. However, our studies demonstrated that the activation of lipolysis does not involve a high affinity binding of the cytosolic lipase to the lipid droplet. Therefore, a change in the kinetic properties of the lipase could be responsible for the enhancement in the lipase activity that is observed when the lipid droplets are phosphorylated with PKA in vitro or when the lipid droplets are isolated from AKH-stimulated insects. This increase in the catalytic activity could be due to an increase in the accessibility of the lipase to the TG molecules. How does the TG-lipase gain access to its substrate? Is the phosphorylation status of Lsdp1 important, perhaps for TG-lipase to specifically dock with the lipid particle? This study suggests that the investigation of the role of the structure and phosphorylation of Lsdp1 in the accessibility of the lipase to TG could provide relevant information to understand the molecular mechanism of activation of lipolysis in insects.

	FOOTNOTES

 
^* This work was supported by National Institutes of Health Grant GM 64677 and the Oklahoma Agricultural Experiment Station. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Biochemistry and Molecular Biology, Oklahoma State University, 246 Noble Research Center, Stillwater, OK 74078. Tel.: 405-744-7505; Fax: 405-744-7799; E-mail:estela{at}biochem.okstate.edu.

¹ The abbreviations used are: TG, triglyceride; AKH, adipokinetic hormone; DG, diglyceride; TLC, thin layer chromatography; HDLp, high density lipophorin; LDLp, low density lipophorin; PKA, protein kinase A; HSL, hormone-sensitive lipase; Peri A, perilipin A; PAT, Peri A, ADRP, and TIP47; MOPS, 4-morpholinepropanesulfonic acid.

	ACKNOWLEDGMENTS

 
We thank Palaniappan Chetty for the MALDI-TOF analysis of Lsdp1 and Hamada Masakazu for rearing the insects.

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