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Proc Natl Acad Sci U S A. Oct 7, 2008; 105(40): 15328–15333.
Published online Oct 1, 2008. doi:  10.1073/pnas.0807833105
PMCID: PMC2563099

A Drosophila orphan G protein-coupled receptor BOSS functions as a glucose-responding receptor: Loss of boss causes abnormal energy metabolism


Glucose, one of the most important nutrients for animals, acts as a regulatory signal that controls the secretion of hormones, such as insulin, by endocrine tissues. However, how organisms respond to extracellular glucose and how glucose controls nutrient homeostasis remain unknown. Here, we show that a putative Drosophila melanogaster G protein-coupled receptor, previously identified as Bride of sevenless (BOSS), responds to extracellular glucose and regulates sugar and lipid metabolism. We found that BOSS was expressed in the fat body, a nutrient-sensing tissue equivalent to mammalian liver and adipose tissues, and in photoreceptor cells. Boss null mutants had small bodies, exhibited abnormal sugar and lipid metabolism (elevated circulating sugar and lipid levels, impaired lipid mobilization to oenocytes), and were sensitive to nutrient deprivation stress. These phenotypes are reminiscent of flies defective in insulin signaling. Consistent with these findings are the observations that boss mutants had reduced PI3K activity and phospho-AKT levels, which indicates that BOSS is required for proper insulin signaling. Because human G protein-coupled receptor 5B and the seven-transmembrane domain of BOSS share the same sequence, our results also have important implications for glucose metabolism in humans. Thus, our study provides insight not only into the basic mechanisms of metabolic regulation but also into the pathobiological basis for diabetes and obesity.

Keywords: energy homeostasis, fat body, insulin signaling

Glucose functions as a cellular fuel or carbon source in most organisms. Sugars and other nutrients such as amino acids and fatty acids can serve as important signaling molecules in complex, nutrient-sensing transduction pathways (1). Glucose triggers intracellular signaling cascades that regulate various aspects of fuel and energy metabolism and influence cell growth, proliferation, and survival. Recent studies have shown that glucose sensors are found in various hypothalamic neurons and endocrine cells (the pancreas and gut), where they function to regulate feeding behavior (2, 3). Furthermore, studies on mammals and Drosophila have suggested that glucose-based metabolism, including neuronal circuits that control feeding, is evolutionarily conserved; thus, Drosophila as a model system provides insights that were not evident from studies of more complex vertebrate systems (4, 5). Although many glucose-sensing systems have been identified in the past few years, details remain elusive about the upstream sensing mechanisms in glucose regulatory pathways, especially whether glucose-sensing receptors exist in cell surface membranes. Drosophila Bride of sevenless (BOSS) is an orphan G protein-coupled receptor (GPCR) membrane protein that was first identified as a ligand for sevenless tyrosine kinase (SEVENLESS), which is involved in eye differentiation in Drosophila melanogaster (6). However, the physiological function of BOSS as a GPCR is completely unknown, because a ligand for BOSS has yet to be identified. We noticed that the bodies of boss-deficient mutants are reduced in size, suggesting that BOSS might be involved in the metabolic regulatory system required for cell growth and survival. In this article, we provide evidence that BOSS has a unique, critical function: a glucose-responding GPCR required for the homeostatic metabolism of glucose and lipids in Drosophila. Consistent with this conclusion is our finding that BOSS is expressed in nutrient-sensing tissue, the fat body, and that BOSS responds to glucose stimulation. In addition, mutants defective in boss exhibit impaired metabolism. Defective boss results in elevated circulating sugar and lipid levels, impaired lipid mobilization, and increased sensitivity to starvation stress. Drosophila insulin receptor/PI3K signaling (Inr/PI3K) plays a central role in coordination of cellular metabolism with nutritional conditions (7). Consistent with the role of Inr/PI3K is our finding that boss-defective mutants have down-regulated insulin signaling activity. Although BOSS was originally identified as a ligand for sevenless in photoreceptor cells and was purported to be involved in differentiation, our results demonstrate that BOSS has an additional critical function: to respond to extracellular glucose levels and regulate energy homeostasis. This finding represents an example of a glucose-responding GPCR in a multicellular organism. Because the GPCRs and signaling pathways for maintaining energy homeostasis are conserved across Drosophila and other species, our study provides insight into nutrient-sensing mechanisms that occur through GPCRs and has important evolutionary, physiological, and clinical implications.


Nutrient-Sensing Tissue Expresses BOSS.

BOSS was first identified as a ligand for sevenless tyrosine kinase, which is involved in eye differentiation in D. melanogaster (Fig. 1A) (6). The N-terminal region of BOSS functions as a SEVENLESS ligand. The seven transmembrane (7TM) and C-terminal regions of BOSS show high sequence identity with the orphan receptor GPRC5B of human (28%) and other species (8, 9) [Fig. 1 A and B and supporting information (SI) Fig. S1]. Interestingly, we noticed that the bodies of boss null mutants (boss1; Fig. 1A) were smaller than those of control flies (HRP-boss) (Fig. 1 C and D).

Fig. 1.
BOSS contains a GPCR motif and is expressed in the fat body. (A) Schematic diagrams of the structures of BOSS, boss1 allele, and vertebrate GPRC5B. GPRC5Bs only have the GPCR motif (7TM regions) of BOSS. The boss1 allele contains a DNA deletion, and thus ...

To identify the roles of BOSS, we fully characterized the tissue distribution of BOSS. It is expressed in many sensory neurons in the developing antenna, leg disks, ocelli, and embryo, in addition to the photoreceptor cells (10). We also examined BOSS expression in both third-instar (L3) larvae and adult flies by using anti-BOSS antibody and found that BOSS was also expressed in the fat body of Drosophila L3 larvae (Fig. 1 E and F). Immunoreactivity was also detected in specific areas of the brain and salivary gland but barely detectable in muscle and gut. This expression profile suggested that BOSS might play a role in the development or physiology of these cells. BOSS is expressed in the fat body, a functional equivalent of mammalian liver and white adipose tissue that senses variations in nutrient levels and coordinates peripheral tissue growth via a humoral mechanism (11, 12). Taken together, these results led us to hypothesize that BOSS may play additional roles as a GPCR in multicellular organisms. Thus, we sought to determine its functions beyond its role as a SEVENLESS ligand.

BOSS Responds to Extracellular Glucose.

A BLAST homology search revealed that BOSS contains a fragment that is homologous to trehalose taste receptor (13) and a putative major facilitator superfamily transporter (CG15221), which transports small molecules such as sugar (data not shown). This finding suggested that the BOSS ligand might be carbohydrate-related. To test our hypothesis that BOSS responds to circulating carbohydrates, we used a reporter gene assay to detect GPCR activation and signaling in response to glucose stimulation. Reporter gene assays are based on the ability of GPCR-mediated secondary messengers such as calcium (Ca2+) or cAMP to activate or inhibit a responsive element placed upstream of a minimal promoter, which in turn regulates the expression of a selected reporter protein. In our experiment, a nuclear factor of activated T cells (NFAT)-luciferase reporter gene assay was introduced to measure Gq-mediated intracellular Ca2+ release. We expressed BOSS in HEK293 cells (Fig. 2A) and then examined changes in intracellular Ca2+ concentrations by measuring luciferase activity. Glucose evoked GPCR signaling in a dose-dependent fashion at 1- to 10-mM concentrations (Fig. 2B), only when glucose levels in the culture medium were eliminated 12 h before the assay. This response was very specific to glucose, and BOSS did not respond to other sugars such as trehalose or sucrose (Fig. 2C). However, we did not detect transient changes in calcium concentrations mediated by BOSS, suggesting that calcium-mediated activation of the NFAT reporter may not be an absolute determinant. Furthermore, glucose had a stimulatory effect on ERK activation in BOSS-expressing HEK293 cells (Fig. 2D). These data demonstrate that BOSS is sensitive to extracellular glucose.

Fig. 2.
BOSS responds to extracellular glucose levels. (A) Expression of myc-tagged BOSS in HEK293 cells as determined by Western blot analysis using anti-Myc antibody. (B) Glucose-induced reporter activity in BOSS-expressing HEK293 cells is concentration-dependent. ...

When GPCRs transduce information provided by extracellular stimuli into cells, the internalization of GPCRs occurs (14). Thus, we next examined whether BOSS expressed in the fat body becomes internalized in response to glucose stimulation. Larvae were fasted for 30 min and then fed with 20% glucose. We observed rapid and excess internalization just 5 min after the start of glucose feeding (Fig. 2Eb). Internalized vesicles disappeared 20 min after glucose stimulation (Fig. 2Ee). This type of internalization was not detectable in fasted larvae (Fig. 2Ea). These results demonstrate that BOSS is required for detecting glucose in vivo. Taken together, these results demonstrate that BOSS functions as a glucose-responding receptor. Further studies, however, are required to elucidate the mechanism of BOSS-mediated signaling.

BOSS Mutant Alters Energy Balance.

Next, we examined biochemical phenotypes of boss1 larvae that do not respond to extracellular glucose. We found that hemolymph sugar (glucose and trehalose) concentrations were elevated in boss1 larvae (Fig. 3A), suggesting that BOSS is involved in the glucose homeostatic system. Because changes in circulating sugar levels are linked to alterations in lipid metabolism (15), we examined triacylglycerol (TAG) (12) levels in boss1 larvae. Hemolymph lipid measurements revealed that boss1 larvae had significantly increased TAG levels (Fig. 3B), indicating that lipid metabolism in these mutants was impaired. Circulating sugar and lipid levels in BOSS expression-rescued larvae (HRP-boss) were reduced relative to those in boss1 larvae and were similar to those of WT controls.

Fig. 3.
Sugar and lipid metabolism is impaired in boss1 larvae. (A) Total circulating sugar (glucose and trehalose) levels in hemolymph from WT, boss1, and HRP-boss L3 larvae. Expression of HRP-boss reversed the hyperglycemia exhibited by boss mutants (n > ...

These findings prompted us to determine whether lipid droplets accumulate in the oenocytes of boss1 larvae. Oenocytes were very recently identified as hepatocyte-like cells that accumulate lipid droplets only during starvation (11). The effects of BOSS on metabolic control were confirmed by using oenocytes as an indicator for lipid mobilization. The oenocytes of boss1 larvae contained lipid droplets, regardless of whether the larvae were fed a standard diet or were deprived of food for 14 h (Fig. 3 C and D). It is important to note that this impaired response was also observed when insulin signaling in the fat body was inhibited by overexpression of a dominant-negative form of Drosophila insulin receptor (dInr) (Fig. 3Cg) (11).

Lipolysis in the Mutant Fat Body Is Enhanced During Starvation Conditions.

TAG is stored predominantly in the fat body as stored energy (16) and is critical for supporting an animal during starvation. Thus, we examined the survival rate of boss1 flies during starvation and assessed TAG levels in those flies. We found that boss1 flies were sensitive to starvation (Fig. 4A). After 48 h of starvation, 62% of WT flies remained viable, whereas only 29% of boss1 flies survived. A time-course analysis of TAG levels during starvation showed that TAG stores dropped more rapidly in boss1 flies than in WT flies (Fig. 4B and Fig. S2). BODIPY staining, which detects neutral lipids, in the fat body of boss1 flies was weaker than that in WT after starvation (Fig. 4B). Quantitative determination of TAG levels of the entire body revealed that the lipid consumption rate was higher in boss1 flies than in WT flies (Fig. S2). These results indicate that the regulatory mechanisms in lipolysis in boss1 flies are not functioning properly.

Fig. 4.
BOSS mutant flies are sensitive to starvation. (A) Survival curves demonstrate starvation sensitivity of boss1 mutant. Experiments were performed in triplicate (n = 60 per genotype). Data are means ± SEM. (B) Starvation-induced TAG consumption ...

Insulin Signaling in the Fat Body Is Suppressed in Boss Mutant Larvae.

Recent extensive studies with Drosophila have shown an essential function of Inr/PI3K signaling in coordinating cellular metabolism with nutritional conditions (7). Impaired Inr/PI3K signaling affects glucose and lipid homeostasis, growth, and even feeding behaviors (17). Eliminating Drosophila insulin-like peptide (DILP) secretion by ablating insulin-producing cells (IPCs) increases hemolymph sugar (18), and overexpression of DILP2 reduces hemolymph sugar levels and increases body weight (19). We observed the same phenotypes, increased hemolymph sugar levels (Fig. 3A) and decreased body weight (Fig. 1 C and D), in boss1 larvae. Furthermore aberrant lipid droplets accumulate in the oenocytes of fed larvae is observed in boss1 and insulin signaling inhibiting larvae (Fig. 3C), indicating that Inr/PI3K pathway activity in boss1 larvae is down-regulated. Therefore, we decided to monitor Inr/PI3K signaling activity in boss1 larvae by using a transgenic fly expressing a GFP-PH domain fusion protein (tGPH) as a reporter (7). tGPH-membrane fluorescence is observed when Inr/PI3K is activated. WT larvae responded to glucose stimulation by exhibiting activated Inr/PI3K signaling in the fat body (Fig. 5A Upper). By contrast, boss1 larvae failed to respond (Fig. 5A Lower). Consistent with this result, the amount of phosphorylated AKT (pAKT; activated form) in boss1 larvae was reduced relatively to that in WT larvae under both fed and fasting conditions (Fig. 5B). These observations support the notion that BOSS affects insulin-signaling activity.

Fig. 5.
Boss affects Inr/PI3K signaling activity. (A) In boss1 larvae, glucose stimulation does not activate the Inr/PI3K signaling pathway in the fat body. Thus, tGPH is not recruited to membranes (Lower). However, in WT larvae, glucose stimulation activates ...

To examine how BOSS signaling affects insulin signaling, we treated the fat body from L3 larvae with insulin, and then examined the membrane localization of tGPH. In the fat bodies of boss1 larvae, we observed increased tGPH membrane localization in response to insulin (Fig. S3A), indicating that Inr/PI3K signaling in the fat body was functional. Treating insulin-stimulated tissues with wortmannin, a specific PI3K inhibitor, abolished tGPH membrane localization. The amount of dilp2, dilp3, and dilp5 mRNA expression was either comparable or modestly increased in boss1 larvae relative to that in WT larvae and flies fed ad libitum. (Fig. S3 B and C). Although further experiments are required to determine how BOSS signaling affects insulin signaling and identify the mechanism underlying these effects, these observations indicate that insulin processing, including insulin secretion and stability, in boss1 larvae is suppressed but insulin signaling in response to DILPs is functional.


Additional Function of BOSS as a Glucose-Responding GPCR.

Our discovery of an additional function of BOSS as a glucose-responding GPCR is an example in multicellular organisms of a glucose-responding GPCR that regulates glucose and lipid metabolism. Sequence similarity between the BOSS 7TM–C-terminal region and vertebrate GPRC5Bs (Fig. 1A and Fig. S1) strongly suggests that the conserved region of BOSS functions as a glucose-responding receptor (Fig. S4). Supporting this suggestion is the observation that both BOSS and GPRC5B expressed in the larval fat body were internalized in response to glucose stimulation (Fig. S5). The same response to glucose stimulation was also observed in GPRC5B-expressing HEK293 cells (data not shown). This type of glucose-dependent internalization was specific to GPRC5B: We did not detect internalization of GPRC5D, which belongs to the same subfamily as GPRC5B (Fig. S5). Although we provided evidence that BOSS responds to glucose through our glucose-dependent receptor-internalization, luciferase reporter assay, and ERK activation results (Fig. 2D), we did not succeed in demonstrating via in vitro assay that glucose binds BOSS directly. Because a constant supply of glucose is essential for cell survival, it would be also very difficult to determine in an in vitro system whether glucose is a specific agonist (ligand) for BOSS. In yeasts, the nutrient-sensing activity of Gpr1, the first GPCR glucose sensor to be identified, was demonstrated through phenotypic analysis of Gpr1 mutants (2022). Thus far, the direct interaction between Gpr1 and glucose has yet to be demonstrated. Similar examples are seen with the taste-receptor Tas1r3 (23). Similar problems have also been encountered in identifying the ligands of deorphanizing GPCRs, such as an amino acid-sensing GPCR (24) and proton-sensing GCPRs (25), even though the agonists of these GPCRs are naturally abundant molecules.

Function of BOSS in Metabolic Regulation.

The mechanisms underlying energy homeostasis in Drosophila and mammals are well conserved: In both, circulating sugars and lipid levels are tightly regulated and depend on insulin signaling and counteracting signals such as glucagon or adrenalin (26). As for insulin-producing tissues, IPCs and pancreatic β-cells have evolved from a common ancestral insulin-producing neuron (27). On the other hand, adipokinetic hormone (AKH), which corresponds to mammalian glucagon and adrenaline, is produced by the corpora cardiaca cells (CCs) of Drosophila (28). Both DILPs and AKH signaling regulate hemolymph sugar levels and lipid metabolism (27, 28). As with the mammalian liver and white adipose tissue, the Drosophila fat body acts as a source of circulating hormones, cytokines, glycogen, and lipids that can influence systemic energy balance and glucose homeostasis (29). Glucose and lipid metabolism systems are impaired in boss mutants, suggesting that BOSS affects the activity of these pathways. We demonstrated that insulin signaling is impaired in boss-defective mutants by using tGPH, an insulin signaling activity indicator (Fig. 5A), and by measuring physiological pAKT levels (Fig. 5B). Furthermore, the phenotypes of boss-defective mutants are similar to those exhibited by flies in which Inr/PI3K signaling is down-regulated: Both types of flies have elevated glucose levels (Fig. 3A), and the oenocytes of both boss-defective and FB>dInRDN mutants accumulate lipids under feeding conditions (Fig. 3C). Because dilps mRNA expression and downstream insulin signaling are not affected by the boss mutation (Fig. S3), we propose that boss-mediated signaling strongly affects insulin processing, including insulin secretion or stability, but not insulin production in vivo. Recently, two DILP2 binding proteins, imaginal morphogenesis protein–Late 2 (lmp-L2) and acid-labile subunit (ALS), were identified as nutritionally controlled suppressors of growth in Drosophila (19, 30), demonstrating that the regulatory mechanisms of insulin signaling are very complicated and elaborate. These proteins form a circulating trimeric complex with DILP2 and control animal growth and carbohydrate and fat metabolism. Further analysis is required to understand precisely how and where BOSS signaling regulates insulin-dependent signaling activities.

Recently, both AKH- and Brummer (BMM) adipose triglyceride lipase–dependent pathways have been reported to be essential for adjusting normal body fat content (31). AKH and BMM signaling pathways control lipolytic rate, and the lack of dual lipolytic control blocks fat mobilization. Although we have not yet examined AKH and BMM signaling activities in the boss mutant fat body, we observed that boss1 mutants had increased hemolymph trehalose levels and sensitivity to starvation stress, indicating that BOSS signaling pathways might also affect AKH and BMM signaling in addition to Inr/PI3K signaling (Fig. S4). BOSS-mediated regulation of AKH and BMM may possibly occur through Inr/PI3K signaling, because the cross-talk between insulin signaling and glucagon signaling occurs in mammals and may also occur in Drosophila, as can be deduced from the projection pattern of IPC axons to CCs (27). Whatever the case, further studies on how BOSS regulates these signaling pathways should provide additional insights into how energy homeostasis is maintained.

Conserved Mechanisms of Glucose-Responding System in Vertebrates.

Glucose-sensing pathways trigger metabolic signaling cascades that regulate various aspects of fuel and energy metabolism and influence cell growth, proliferation, and survival. Current knowledge about the upstream sensing mechanisms in glucose regulatory pathways is limited. In mammals, a sugar-sensing GPCR similar to the yeast GPCR (20, 22) that acts through the cAMP pathway has been proposed to exist in intestinal epithelial cells (32). Furthermore, hypothalamic orexin neurons have been proposed to harbor cell surface receptors that respond to extracellular glucose (33). However, the precise characteristics of such receptors remain unclear. Because Drosophila shares most of the same basic metabolic functions found in vertebrates and also possesses BOSS orthologous GPCRs (GPRC5Bs), our identification of a glucose-responding membrane receptor in Drosophila can provide another clue toward deciphering glucose-sensing mechanisms in vertebrates.

Materials and Methods

Drosophila Strains.

We used the following nine strains of Drosophila: w1118 (WT; used as controls in most of our experiments); w; P[w+, gen.HRP-boss]; boss1 Sb/boss1 TM6B (gift from H. Kramer, University of Texas Southwestern Medical Center, Dallas, TX; used as boss-rescued strain); tGPH [a pleckstrin homology domain–GFP (PH-GFP; GPH) fusion gene was placed under control of the Drosophila β-tubulin promoter] (7); FB-Gal4 (34) (gift from R. Kuhnlein, Max-Planck-Institut for Biophisikalische Chemie, Göttingen, Germany); ppl-Gal4 (35) (kind gift from M.J. Pankratz, Institut for Genetik, Dresden, Germany); UAS-dInRDN (UAS-InRK1409A) (36) (from the Bloomington Stock Center); and UAS-boss (boss cDNA was cloned into the pUAST vector). The boss1 allele is a null allele that contains an additional 24 aa plus a deletion in which the codon for amino acid 45 differs from that of the WT but still encodes alanine (6).

Boss cDNA was obtained from Berkeley Drosophila Genome Project–Drosophila Genome Collection (BDGP-DGC) clones containing full-length boss cDNA (GH10049). BDGP-DGC clones and UAS-GPRC5B and UAS-GPRC5D (mouse GPRC5B cDNA and GPRC5D cDNA, respectively) were cloned into pUAST vectors. GFP (Venus) was fused to the C termini of GPRC5B and GPRC5D.

Cell Culture, Plasmids, and Transfection.

HEK293 Flp-In cells were obtained from Invitrogen. Cells were maintained in DMEM supplemented with 10% FBS and cultured in a humidified 5% CO2 incubator.

The N-terminal end of the boss-coding region (amplified by PCR from BDGP-DGC clones; GH10049) was fused to an Igκ leader sequence to facilitate plasma-membrane localization, and the C-terminal end was fused to a Myc-epitope sequence. The tagged boss was then cloned into a pcDNA5/FRT vector, which was designated pBOSS-myc. The plasmid pOG44 expressing Flp recombinase was obtained from Invitrogen. The luciferase reporter plasmid pNFAT-luc was purchased from Stratagene. The GFP-expression vector pEGFP-N1 was purchased from Clontech.

Transient or stable DNA transfection was performed by using Fugene 6 (Roche) transfection reagent according to the manufacturer's instructions.

Luciferase Assay.

HEK293 cells were plated onto 24-well collagen-coated plates (IWAKI) the day before transfection. Cells were cotransfected with 1 μg of pBOSS-myc, 1 μg of pNFAT-luc, and 0.1 μg of pEGFP-N1 per well. Mock control cells were transfected with the same amount of pcDNA/FRT vector instead of pBOSS-myc and with pNFAT-luc and pEGFP-N1. Twelve hours after transfection, cells were cultured in glucose-free DMEM containing 0.1% fatty acid-free BSA (Sigma), and then stimulated with various concentrations of glucose for 12 h. To measure luciferase activity, we lysed the cells in Glo lysis buffer (Promega) and incubated them in Steady Glo luciferase substrate (Promega). Transfection efficiency was corrected by means of EGFP fluorescence intensity. Luciferase activity was measured with a POWERSCAN HT microplate fluorescence reader (Sumitomo).

Phospho-ERK Assay.

BOSS was stably expressed in HEK293 Flp-In cells. Polyclonal stable cells expressing BOSS were selected in culture medium containing 0.1 mg/ml hygromycin (Invitrogen). After selection, BOSS expression was confirmed by Western blotting using anti-myc antibody (Cell Signaling), as shown in Fig. 2A. Before stimulation, cells in 24-well collagen-coated plates were starved for 12 h in glucose-free medium. After stimulation with 20 mM glucose, cells were lysed in RIPA buffer (20 mM Hepes, 150 mM NaCl, 1% Triton X-100, 0.1% SDS, 0.25% deoxycholate, and 2 mM EDTA) supplemented with protease and phosphatase inhibitor cocktails (Roche). Equal amounts of protein extract were separated in 10% Tris-glycine polyacrylamide gels and transferred onto PVDF membranes for Western blotting. Phosphorylated ERK1/2 and total ERK1/2 were detected with rabbit polyclonal anti-phospho-p42/44 MAPK and anti-p42/44 MAPK antibodies (Cell Signaling), respectively. Chemiluminescence detection was performed with Immobilon Western Detection Reagent (Millipore).

Glucose and Trehalose Measurements.

Three to four L3 larvae were washed twice with distilled water and blotted dry. The cuticle was torn away to allow hemolymph to bleed out, and 0.5 μl of hemolymph was rapidly withdrawn and mixed with 14.5 μl of PBS. The diluted hemolymph was added to 100 μl of Infinity Glucose Reagent, adjusted to pH 6.8 (Thermo DMA), incubated at 37°C for 16 h, and measured by using quantitative NADH fluorescence detection (at an excitation wavelength of 375 nm and an emission wavelength of 465 nm) with a fluorescence plate reader. To measure trehalose, 10 μl of pig kidney trehalose (Sigma) was added to 5 ml of Infinity Glucose Reagent. Standard curves were generated for glucose and trehalose concentrations for each trial; the assay was linear over a range of 0–1,000 mg/ml.


Tissues were dissected in PBS, fixed in 4% paraformaldehyde for 25 min at room temperature, and washed four times in PBS/0.1% Triton-X (PBST). Tissues were incubated with primary antibodies (1:100, anti-BOSS antibody, gift from H. Kramer; 1:500, anti-GFP antibody, Aves Labs) and subsequently with FITC-tagged secondary antibodies, and the resulting fluorescence signals were examined under a confocal microscope LMS5 PASCAL (Zeiss). Antibodies were diluted in PBST containing either 5% normal goat serum or 5% donkey serum.

Glucose Stimulus Assay.

L3 larvae fed with normal food were cultured in H2O for 30 min, and then they were cultured in 20% glucose for 5, 15, and 30 min. BOSS or GPRC5B was overexpresed in the fat body by using the FB-Gal4 driver. To detect Inr/PI3K signaling activity, we used tGPH larvae for this assay [control (w;tGPH) and boss mutant (w;tGPH;boss1)]. Fat bodies were fixed and stained with anti-boss antibody or anti-GFP antibody to visualize the cellular localization of BOSS, GPRC5B, and tGPH, which was assessed with a confocal microscope (LMS5 PASCAL).

TAG Assay.

To measure circulating TAG, we used the enzymatic method as described (12). Batches of 20 larvae or 10 flies of the desired genotypes were homogenized in 0.05% Tween 20 in the presence of protease inhibitor (Sigma inhibitor mixture) in H2O on ice. The homogenates were spun at 2300 × g on a tabletop centrifuge for 1 min, and 500 μl of supernatant was transferred to a fresh tube void of debris. The supernatant was then spun at 17800 × g for 3 min on a tabletop centrifuge at 4°C. To measure circulating TAG, 80 μl of the resulting supernatant was combined with 1 ml of Triglyceride Reagent (Sigma) and incubated for 10 min at 37°C. The OD520 nm was measured and compared to a standard curve. To measure protein levels, 100 μl of the final supernatant was combined with 700 μl of H2O and 200 μl of Bio-Rad Protein Assay Reagent and incubated for 3 min at room temperature, and the OD595 nm was measured and compared with a standard curve.

Oil-Red-O and 4,4-Difluoro-1,3,5,7,8-pentamethyl-4-Bora-3α,4α-Diaza-s-Indacene Propionic Acid (BODIPY) Staining.

Adult flies or dissected larvae were fixed in 4% paraformaldehyde for 25 min at room temperature and washed four times in PBS. Oil-Red-O staining was done as described (11). To visualize neutral lipids, we stained the fixed fat bodies with BODIPY 493/503 (Molecular Probes). BODIPY 493/503 was dissolved in ethanol at 1 mg/ml, and then added to PBS to a final concentration of 2 μg/ml. BODIPY in PBS was applied to tissue samples. After incubation for 1 h, tissues were washed three times with PBS.

Starvation Assay.

Ten male flies of each genotype, 4 days of age, were transferred to vials provided with water supply only. Mortality rates were determined by regularly counting the number of dead flies. Dead flies lacked a sit-up response. We then plotted average survival rate values and the corresponding standard deviations of a representative experiment.

Western Blotting.

Protein extracts from 10 L3 larvae were separated on 10% Tris-glycine polyacrylamide gels and transferred onto PVDF membranes for Western blotting. Phospho-AKT was detected with anti-pAKT (1:500) (9271; Cell Signaling Technology). Chemiluminescence detection was performed by using Immobilon Western Detection Reagent (Millipore).


Total RNA was extracted from L3 larvae and 4-day-old adult flies, and reverse-transcription reactions were performed by using SuperScript-II enzyme (Invitrogen). Semiquantitative PCR was performed on aliquots from these reaction mixtures. Actin-5C was used as an internal loading control. See Table S1 for primers used.

Additional Methods.

See SI Text for more information on methods used.

Supplementary Material

Supporting Information:


We thank Drs. H. Kamiguchi, A. Miyawaki, and A. Taguchi for helpful advice and Y. Nagatuka, T. Sano, Y. Shinoda, E. Oshima, T. Shimizu, and N. Õashi for technical assistance.


The authors declare no conflict of interest.

This article contains supporting information online at www.pnas.org/cgi/content/full/0807833105/DCSupplemental.


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