Heart- and muscle-derived signaling system dependent on MED13 and Wingless controls obesity in Drosophila

Significance

Obesity is a major health epidemic and develops as a result of imbalanced energy homeostasis. Previously, we reported that cardiac expression of MED13, a subunit of the Mediator complex, controlled systemic energy homeostasis in mice such that increased or decreased expression of MED13 caused leanness or obesity, respectively. Here, we report that MED13 also acts within muscle of Drosophila to control obesity. The secreted peptide Wingless acts as a downstream effector of MED13 to mediate cross-talk with adipose tissue and suppress obesity. Our work reveals a conserved signaling system in muscle in which MED13 and Wingless act as key controllers of obesity.

Abstract

Obesity develops in response to an imbalance of energy homeostasis and whole-body metabolism. Muscle plays a central role in the control of energy homeostasis through consumption of energy and signaling to adipose tissue. We reported previously that MED13, a subunit of the Mediator complex, acts in the heart to control obesity in mice. To further explore the generality and mechanistic basis of this observation, we investigated the potential influence of MED13 expression in heart and muscle on the susceptibility of Drosophila to obesity. Here, we show that heart/muscle-specific knockdown of MED13 or MED12, another Mediator subunit, increases susceptibility to obesity in adult flies. To identify possible muscle-secreted obesity regulators, we performed an RNAi-based genetic screen of 150 genes that encode secreted proteins and found that Wingless inhibition also caused obesity. Consistent with these findings, muscle-specific inhibition of Armadillo, the downstream transcriptional effector of the Wingless pathway, also evoked an obese phenotype in flies. Epistasis experiments further demonstrated that Wingless functions downstream of MED13 within a muscle-regulatory pathway. Together, these findings reveal an intertissue signaling system in which Wingless acts as an effector of MED13 in heart and muscle and suggest that Wingless-mediated cross-talk between striated muscle and adipose tissue controls obesity in Drosophila. This signaling system appears to represent an ancestral mechanism for the control of systemic energy homeostasis.

Obesity is a systemic disorder caused by an energy imbalance in which energy input exceeds energy utilization, resulting in accumulation of excess body fat. Muscle plays a central role in systemic energy homeostasis by consuming nutrients and signaling in an endocrine manner to other tissues (1–3). Thus, there has been intense interest in identifying secreted factors from muscle that modulate the function of adipose tissue.

Previously, we reported that cardiac deletion of MED13, a subunit of the Mediator complex, increases susceptibility to obesity in mice whereas cardiac overexpression of MED13 confers a lean phenotype (4), revealing an unforeseen function of the heart as a systemic regulator of energy homeostasis. The Mediator is a conserved multisubunit complex that mediates the interaction between RNA polymerase II and transcription factors and therefore governs transcription in all eukaryotes (5). MED13 and MED12 are among four subunits of the auxiliary kinase module, which confers additional regulatory functions to the Mediator complex (6). Expression profiling of yeast mutants or gene-depleted Drosophila cell lines revealed a close correlation between the gene-expression programs controlled by MED13 and MED12, suggesting their concerted actions in gene regulation (7, 8).

Drosophila provides a powerful model system for the genetic analysis of obesity (9–11). The processes that regulate energy homeostasis, such as energy storage and mobilization of fat in adipose tissue of the fat body, as well as the genetic pathways governing such functions, are conserved in flies (12). Perturbations of such pathways in flies have been shown to generate phenotypes relevant to human diseases, including obesity (13–15). Furthermore, Drosophila serves as a model system to understand systemic effects of interorgan cross-talk via secreted molecules (3, 16).

Here, we show that Drosophila muscle modulates obesity through the function of MED13 in the context of the Wingless (Wg) signaling pathway. In Drosophila, MED13 and MED12 are encoded by skuld (skd) and kohtalo (kto), respectively (17). Muscle-specific knockdown of skd or kto increases fat body mass and triglyceride accumulation in adult flies. We describe a genetic screen for muscle-secreted obesity regulators, which revealed Wg as a muscle-derived suppressor of obesity. Similarly, inhibition of Armadillo (Arm), the Drosophila β-catenin ortholog and transcriptional effector of Wg signaling, suppresses obesity. We also identify functional relationships between skd and the Wg pathway in which a skd-null mutation dominantly enhances the muscle phenotype resulting from arm knockdown, and wg acts downstream of skd in the regulation of fat accumulation. Our findings indicate that Wg acts as an effector of MED13 function in muscle to suppress obesity in Drosophila.

Results

Loss of MED13 Function in Muscle Increases Susceptibility to Obesity in Drosophila.

Based on our observation that cardiac deletion of MED13 confers an obese phenotype and that cardiac-specific overexpression of MED13 prevents obesity in mice (4), we examined whether muscle expression of MED13, encoded by skd, regulates fat accumulation in Drosophila. We performed RNAi-mediated knockdown experiments using the UAS/Gal4 system and expressed UAS-RNAi targeting skd mRNA with the Mef2-Gal4 driver, which directs the expression of UAS constructs in somatic, cardiac, and visceral muscle tissues (18–21). By 3 wk of age, we observed increased abdominal fat bodies in adult Mef2>skd RNAi flies (Fig. 1A). Lipid droplets in the fat body cells were also enlarged as seen by Nile Red stain (Fig. 1B). Consistently, total triglyceride amounts were significantly increased in those flies (Fig. 1C). Using a different fly muscle-specific driver, Mhc-Gal4 (22), to target skd mRNA, we observed that Mhc>skd RNAi flies also displayed increased total triglycerides (Fig. 1D).

Fig. 1.

MED13 expression in muscle regulates obesity. (A) Abdominal fat bodies of 3-wk-old adult females expressing either skd RNAi or gfp (control) with Mef2-Gal4. (B) Confocal images of the abdominal fat body from Mef2>skd RNAi or Mef2>gfp (control) flies stained with Nile Red (red) and Phalloidin (green). (Scale bar: 50 μm.) (C–E) Relative triglyceride amounts of adult females with muscle-specific knockdown of skd, kto, or luciferase (control) using Mef2-Gal4 (C), Mhc-Gal4 (D), or Tin-Gal4 (E). Error bar, SEM; *P < 0.05; **P < 0.01; ***P < 0.001. (F) Survival curves showing resistance of adult females with indicated genotypes under starvation conditions. Flies were maintained under normal conditions for 3 wk and then moved to 1% agar. Mef2>skd RNAi (median survival, 108 h, n = 120); Mef2>kto RNAi (median survival, 96 h, n = 80); Control, Oregon R (median survival, 72 h, n = 72). (G) Survival curves showing overall lifespan of females with indicated genotypes. Mef2>skd RNAi (median survival, 81 d, n = 177); Mef2>kto RNAi (median survival, 81 d, n = 149); Control, Oregon R (median survival, 81 d, n = 109).

To test whether perturbation of MED13 function specifically in the fly heart modulates total body triglycerides, skd was knocked down using the heart-specific Tin-Gal4 (23, 24). Tin>skd RNAi flies also showed a significant increase in total triglycerides (Fig. 1E). We also tested whether muscle-specific knockdown of kto, which encodes MED12, affected fat accumulation. Indeed, flies with RNAi knockdown of kto in muscle or heart using Mef2-Gal4, Mhc-Gal4, or Tin-Gal4 displayed increased total body triglyceride levels similar to the effect of skd knockdown (Fig. 1 C–E).

Resistance to starvation is a characteristic of obese flies (25). We tested whether skd or kto knockdown in muscle conferred resistance to starvation. Three-week-old flies with Mef2-Gal4 driven knockdown of skd or kto survived substantially longer under starvation conditions of 1% agar (Fig. 1F). However, their overall lifespans under normal conditions did not change (Fig. 1G).

To confirm that skd or kto knockdown using the RNAi lines indeed inhibited their expression, we tested the effects in the fly eye, where skd or kto loss-of-function has been shown to prevent photoreceptor differentiation (26), resulting in small eye phenotypes in adults (27). We expressed the RNAi lines using the eye-specific eyeless-Gal4 and GMR-Gal4 and observed small eye phenotypes from both skd and kto knockdown flies, similar to the documented loss-of-function mutant eye phenotype (Fig. S1), confirming effective RNAi knockdown.

We tested whether the obese phenotype associated with skd or kto knockdown in muscle was age-dependent. Flies with muscle-specific knockdown of skd or kto showed no obvious changes in triglycerides 2 wk after eclosion, but a substantial increase was observed by 3 wk and 4 wk of age. By 5 wk of age, fat accumulation reached a maximum level irrespective of genotype (Fig. 2A).

Fig. 2.

Flies with MED13 or MED12 knockdown display increased susceptibility to obesity. (A) Age-dependent changes in total triglyceride levels. Mef2-Gal4 was used to drive knockdown of skd or kto. Control 1, Mef2-Gal4 alone; control 2, Mef2>gfp. (B) Effects of high-fat diet on total triglyceride amounts in flies with the indicated genotypes. Twelve-day-old females grown in normal food were transferred to normal (Left) or high-fat (Right) food and maintained for 3 d. With normal food, Mef2>skd RNAi and Mef2>kto RNAi increased triglycerides by 22% and 23% on average, respectively, but the differences were statistically insignificant (P > 0.05). With high-fat food, Mef2>skd RNAi and Mef2>kto RNAi caused an increase of triglycerides in flies to 57% and 33% on average, respectively. Control harbors Mef2>gfp. Error bar, SEM; NS, not significant; *P < 0.05; **P < 0.01.

We next tested whether flies with muscle-specific skd or kto knockdown were more susceptible to triglyceride accumulation on a high-fat diet, which contains coconut oil as the source of saturated fat (28). Newly eclosed flies with Mef2-Gal4–driven knockdown of skd or kto were fed normal food for 12 d and then moved to either fresh normal food or high-fat food containing 30% coconut oil. Three days later, the flies fed high-fat food increased triglycerides substantially compared with the control flies under the same growth condition whereas those fed normal food did not (Fig. 2B).

An RNAi Screen Identifies Muscle-Secreted Proteins Controlling Obesity.

We hypothesized that the obese phenotype resulting from muscle-specific knockdown of skd or kto was mediated by extracellular factors secreted by muscle cells. To test this hypothesis, we performed an RNAi-based genetic screen using Mef2-Gal4 to identify muscle-secreted proteins that, when inhibited, caused an obese phenotype, similar to what was observed with skd or kto knockdown in muscle. We analyzed 182 RNAi lines targeting 150 genes that encode secreted proteins. Relative total triglyceride levels of these flies at 4 wk of age were measured in comparison with the average of six controls (Table S1). Because skd knockdown using either Mef2-Gal4 or Mhc-Gal4 caused the fat accumulation phenotype (Fig. 1 C and D), we performed the knockdown screen again using the same set of RNAi lines expressed with Mhc-Gal4 (Table S2). We identified six RNAi lines that increased total triglyceride levels greater than 60% as a result of knockdown with both Mef2-Gal4 and Mhc-Gal4 (Table 1).

Table 1.

Six RNAi lines that increase triglyceride amounts >60% from Mef2-Gal4 and Mhc-Gal4 screens

Gene name	Annotation symbol	BDSC no.	Mef2 > RNAi*	Mhc > RNAi*
Diptericin B	CG10794	28975	1.84	2.10
Angiotensin converting enzyme	CG8827	36749	1.76	1.93
SIfamide	CG33527	29428	1.74	2.26
Wingless	CG4889	33902	1.69	1.72
Insulin-like peptide 4	CG6736	33682	1.65	2.13
Unpaired 3	CG33542	32859	1.63	1.63

BDSC, Bloomington Drosophila Stock Center.

^*Indicated values are relative triglyceride amounts (triglyceride/protein) compared with controls.

Wg Signaling in Muscle Suppresses Obesity.

We identified wg from the genetic screen described above (Table 1) and confirmed the effect of wg using two independent RNAi lines. Flies expressing either wg RNAi with Mef2-Gal4 showed increased abdominal fat body mass (Fig. 3A). The lipid droplets therein were enlarged (Fig. 3B), and total triglyceride amounts of the flies were also increased (Fig. 3C). To confirm the effect of wg knockdown in muscle on total triglycerides and to test the heart-specific effect of wg knockdown, we used Mhc-Gal4 and Tin-Gal4, respectively, to express wg RNAi and found that knockdown of wg with either of the drivers resulted in a substantial increase of body triglyceride content (Fig. 3 D and E).

Fig. 3.

The Wg signal in muscle regulates obesity. (A) Abdominal fat bodies of adult females expressing luciferase (luc) RNAi (control), wg cDNA, wg RNAi, arm^S10 cDNA (constitutively active), arm^S2 cDNA (wild-type), or arm RNAi with Mef2-Gal4. (B) Confocal images of adult abdominal fat bodies stained with Nile Red (red) and Phalloidin (green). Genotypes are as indicated above (A). (Scale bar: 20 μm.) (C–E) Effects of muscle-specific knockdown or overexpression of Wg or Arm on relative triglyceride amounts in adult females using Mef2-Gal4 (C), Mhc-Gal4 (D), or Tin-Gal4 (E). Control was luciferase RNAi. Error bar, SEM; *P < 0.05; **P < 0.01; ***P < 0.001.

Consistent with the results from wg knockdown experiments, wg overexpression in muscle decreased fat accumulation. Two independent UAS-wg cDNA lines were tested with Mef2-Gal4. These flies showed a decrease of abdominal fat body mass and lipid droplet size (Fig. 3 A and B). Total triglyceride levels in those flies were also decreased significantly (Fig. 3C). Reduced fat accumulation was also observed in flies overexpressing wg with Mhc-Gal4 or Tin-Gal4 (Fig. 3 D and E).

To investigate whether autonomous Wg signaling activity in muscle has a role in regulating obesity, we tested the effect on fat accumulation of arm, encoding the transcriptional effector of Wg signaling, in muscle. The expression of arm RNAi using Mef2-Gal4 resulted in increased abdominal fat bodies containing enlarged lipid droplets (Fig. 3 A and B). Total triglyceride levels were also increased by muscle-specific knockdown of arm (Fig. 3C). In addition, using Mhc-Gal4 and Tin-Gal4 to knock down arm, we observed an increase in total triglyceride levels (Fig. 3 D and E). Conversely, muscle-specific overexpression of arm either as a wild-type (arm^S2) or a constitutively active (arm^S10) form decreased abdominal fat body mass, lipid droplets in the fat body, and total triglyceride amounts (Fig. 3).

Functional Interaction Between skd and Arm in Muscle.

To test the functional interaction of the Wg pathway and MED13 in muscle, we performed genetic-interaction experiments between arm and skd. Flies with arm knockdown using Mef2-Gal4 are viable without morphological defects. skd^T606 is a null allele that is homozygous lethal (24), but skd^T606 heterozygotes are viable without morphological defects. However, arm knockdown using Mef2-Gal4 in a skd^T606 heterozygous (skd^−/+) background caused complete lethality, which was fully penetrant. To better understand the nature of this lethality, the somatic muscle structure of the embryos was examined. Embryos expressing luciferase RNAi in muscle in a skd^+/+ or skd^−/+ background maintained intact muscle patterns (Fig. 4 A and B). A portion of embryos expressing arm RNAi in muscle in a skd^+/+ background had intact muscle patterns (Fig. 4C) whereas other embryos with the same genotype displayed patterning defects in the somatic musculature as exemplified by defects in lateral transverse (LT) and dorsal muscles (Fig. 4C′). However, all observed embryos expressing arm RNAi using Mef2-Gal4 in the skd^−/+ background had patterning defects in their somatic musculature (Fig. 4 D and D′).

Fig. 4.

Genetic interaction between MED13 and Arm in muscle. Embryos at St. 16 were immunostained with anti-Mhc antibody and shown laterally with the orientation of dorsal up and anterior right. skd− is skd^T606. Embryos in A, B, and C display normal embryonic musculature whereas embryos in C′, D, and D′ have defects in their musculature, some of which are indicated with red asterisks for dorsal muscle defects and dotted boxes highlighting normal (A, B, and C) or abnormal (C′, D, and D′) patterns of LT muscles 1–4. LT, lateral transverse; SBM, segment border muscle.

The Epistatic Relationship of Wg and MED13 in Muscle for Obesity Control.

Our finding that overexpression of wg in muscle caused a lean phenotype (Fig. 3) whereas skd knockdown in muscle caused an obese phenotype (Fig. 1), as well as the functional interaction between skd and arm (Fig. 4), raised the possibility that wg and skd act within a linear pathway in muscle to regulate obesity. To address this possibility, we tested the epistatic relationship between wg and skd. Flies expressing either skd RNAi or wg cDNA or both with Mef2-Gal4 were grown for 4–4.5 wk, and their total triglyceride levels were compared. Total triglyceride levels of flies with both skd knockdown and wg overexpression were significantly decreased compared with those of flies with skd knockdown, but indistinguishable from those of flies with wg overexpression alone (Fig. 5A). We also performed the same experiments using Mhc-Gal4 and Tin-Gal4 and obtained consistent results (Fig. 5 B and C). These data strongly support the conclusion that muscle-secreted Wg acts as a downstream effector of skd function in muscle to suppress fat deposition in the fat body.

Fig. 5.

Wg functions downstream of MED13 in muscle to regulate obesity. (A–C) Epistatic relationship between wg and skd shown with relative triglyceride amounts of 4- to 4.5-wk-old adult females with the expression of wg cDNA or skd RNAi or both in muscle using Mef2-Gal4 (A), Mhc-Gal4 (B), or Tin-Gal4 (C). Error bar, SEM; NS, not significant; **P < 0.01; ***P < 0.001. (D) Images of third instar larvae with Wg overexpression using fat body-specific Dcg-Gal4. Control was Dcg-Gal4 alone. Arrows indicate abdominal region where fat bodies are severely reduced.

Finally, we tested the effect of Wg activation in the fat body. When wg was overexpressed using the fat body-specific Dcg-Gal4 (29, 30), it caused lethality at the pupal stage and severe reduction of fat body mass in the abdominal region of third instar larvae (Fig. 5D). These findings support a model in which muscle-secreted Wg acts on the fat body to inhibit obesity (Fig. 6 and Discussion).

Fig. 6.

A model of muscle-derived signaling via MED13 and Wg for obesity control. Expression of MED13 or Wg in muscle suppresses fat accumulation in the fat body, and Wg acts downstream of MED13. Muscle-secreted Wg activates the Wg signal in both muscle and fat body.

Discussion

Our results reveal a role of muscle in systemic regulation of obesity via the function of MED13 in Drosophila. We performed a genetic screen and identified muscle-secreted obesity-regulating factors, including Wg, and demonstrated that Wg signaling in muscle is necessary and sufficient to suppress obesity. Furthermore, we showed that a skd-null mutation dominantly enhances the arm phenotype in muscle and that wg is epistatic to skd, suggesting that Wg is a downstream effector of MED13 in muscle.

Our results reveal that MED13 in Drosophila muscle functions to suppress obesity based on several criteria, such as histology, measurement of whole-body triglycerides, tolerance to starvation stress, and susceptibility to high-fat diet. Similarly, muscle-specific knockdown of MED12 also increased fat accumulation, suggesting that MED12 and MED13 function similarly in the control of fat deposition in Drosophila. Our finding that MED12 and MED13 modulate energy homeostasis adds to a growing number of examples in which components of the kinase module of the Mediator complex influence metabolic signaling on an organismal level. For example, the other two components of the kinase module, Cyclin-dependent kinase 8 and Cyclin C, have also been reported as negative regulators of fat accumulation in flies and mice (31). Our finding that the activity of MED13 in cardiac muscle regulates fat accumulation in Drosophila is consistent with our earlier observation with mice (4) and suggests that the function of cardiac MED13 in systemic regulation of fat storage represents an ancestral mechanism conserved in metazoans. Although it seems most likely that the effect of MED13 on obesity is mediated by overall changes in metabolism, it is also conceivable that changes in feeding behavior contribute to the obesity phenotypes we observed.

Knockdown of MED12 and MED13 using drivers that are active specifically in the heart using Tin-Gal4 or generally in all muscles using Mef2-Gal4 or Mhc-Gal4 commonly evoked comparable obesity phenotypes. Thus, we conclude that MED12 and MED13 can control metabolic signaling from the heart, consistent with our prior conclusions regarding the functions of MED13 in the mouse heart. However, these Gal4 drivers do not enable us to reach conclusions regarding the specific role of somatic or visceral muscle in this signaling process because Mhc-Gal4 and Mef2-Gal4 are active in diverse muscle-cell types. Given that MED12 and MED13 are ubiquitously expressed, it is possible that they also act in nonmuscle tissues to regulate metabolic homeostasis.

We hypothesized that muscle-secreted factors mediate the function of MED13 in Drosophila muscle to suppress systemic fat accumulation. To identify such factors, we screened for muscle-secreted obesity-regulating proteins using two different muscle drivers, Mef2-Gal4 and Mhc-Gal4. We identified six genes that increased fat accumulation of flies in both screens by >60%, including the genes encoding (i) an antimicrobial peptide, Diptericin B (32); (ii) a Drosophila homolog of Angiotensin converting enzyme (33); (iii) a G protein-coupled receptor ligand SIFamide (34); (iv) one of seven Drosophila Insulin/IGF homologs, Insulin-like peptide 4 (35); (v) a JAK/STAT signaling ligand, Unpaired 3 (36); and (vi) Wg. Interestingly, it has been shown recently that MED13 and MED12 are required for the expression of Diptericin B in response to Immune Deficiency (IMD) pathway activation (8), suggestive of additional regulatory functions of MED13 and the genes identified from our screens beyond obesity control.

We demonstrated that Wg and its autonomous signaling activity, controlled by Arm, in muscle are necessary and sufficient for systemic regulation of obesity in vivo. Previously, the correlation between obesity and the expression of genes involved in the Wnt signaling pathway in heart has been raised from transcriptome analyses using heart biopsies from obese patients (37). Similarly, correlations between obesity and differential expression of genes for Wnt signaling, as well as genes for insulin sensitivity and myogenic capacity, were also found in skeletal-muscle samples from obese rats (38). These findings suggest that Wg signaling activity in muscle serves as an intrinsic rheostat for obesity control.

Muscle-specific arm knockdown caused partial-patterning defects in the embryonic musculature, and a skd-null allele dominantly enhanced this phenotype to complete lethality. Given the central role of Arm in Wg target gene expression, our findings are consistent with the established function of wg in the development of mesoderm and the embryonic musculature (39). Our findings reveal a close functional connection between MED13 and Arm, suggestive of the role of MED13 in Wg target gene expression. In fact, in the developing Drosophila eye and wing, MED13 and MED12 are essential for Wg target gene expression, and the MED13/MED12 complex physically interacts with Pygopus, a component of the Wg transcriptional complex (40). Furthermore, MED12 hypomorphic mutant mice are embryonic lethal with impaired expression of Wnt targets (41). Therefore, our genetic interaction data along with these previous reports suggest that MED13 is a general component of the canonical Wg/Wnt pathway.

Our epistasis experiments indicate that muscle-secreted Wg functions downstream of MED13 in muscle to suppress obesity. Because both wg and arm in muscle are crucial for obesity regulation, one function of muscle-secreted Wg might be to act on muscle. Accordingly, the nonautonomous function of Wg to suppress obesity may occur through autonomous Wg signal activity in muscle. However, if MED13 functions at the level of transcriptional control of Wg target genes and the sole function of muscle-secreted Wg ligand is to activate the Wg signal “in” muscle, Wg should be upstream of MED13, which is contrary to our epistasis studies. Based on our data, it stands to reason that muscle-secreted Wg should also act directly on a tissue other than muscle for its nonautonomous effect. If so, which tissue may be the target? Ectopic expression of Wg using a fat body-specific Dcg-Gal4 decreased larval abdominal fat body mass, which demonstrates the role of Wg signaling in the fat body for fat-mass regulation. Similarly, in mammals, autonomous activation of the Wnt pathway in adipose tissue decreases fat mass. Wnt signaling blocks mammalian adipogenesis in vitro (42), and, in mice, activation of the canonical Wnt pathway in adipocytes by ectopic expression of Wnt10b, a Wnt ligand, inhibits obesity (43, 44). Furthermore, autonomous activation of the Wnt pathway in adipose progenitors with constitutively active β-catenin expression decreases fat mass (45). Therefore, the reduced fat mass in Dcg > wg larvae indicates that autonomous Wg signaling activity in the fat body serves as a regulator of fat mass. Considered together with our data showing that muscle-secreted Wg contributes to nonautonomous regulation of adiposity in vivo, we conclude that muscle serves as a source of Wg to regulate adiposity by modulating Wg signaling activity in fat body. However, we cannot rule out the possibility that the systemic effect of Wg from muscle is mediated through an alternative tissue, such as nervous system (46).

Wg acts on short- and long-range targets. Wg is highly hydrophobic and has been shown to diffuse through the extracellular space and act on long-range targets by associating with solubilizing molecules such as lipoprotein particles and Secreted Wg-interacting molecule (47, 48). Furthermore, Wnt-1 has been identified in serum, and decreased Wnt-1 levels in serum correlate with premature myocardial infarction and metabolic syndrome (49), suggesting that Wg may act on remote organs as an endocrine factor. Therefore, we propose a model in which muscle-secreted Wg is a downstream effector of MED13 and acts both to activate the signal in muscle and to act on the fat body ultimately to achieve systemic inhibition of obesity.

Materials and Methods

Fly Stocks and Genetics.

Fly stocks used are described in SI Materials and Methods. Flies were maintained in vials with Cornmeal-Molasses-Yeast medium except for experiments using high-fat food, which was 30% (wt/vol) coconut oil (MP Biomedicals) added to normal food, as described (28). Detailed procedures of fly genetics are available in SI Materials and Methods.

Triglyceride Assay.

Triglyceride assay was performed as described with modifications (30) and is available in SI Materials and Methods.

Immunohistochemistry.

Antibodies used were anti-Mhc (a gift of Bruce Paterson, National Cancer Institute, Bethesda) and anti-GFP (Torrey Pines Laboratories). Embryos were immunostained as described (50).

Nile Red Staining.

To visualize adult abdominal fat bodies, a vertical incision was made along the ventral abdomen of female flies. Other internal organs were removed and fixed in 4% paraformaldehyde for 15 min. Images were taken using a Zeiss AxioCam. Fixed abdomens were stained with Nile Red (Fluka) at a concentration of 10 μg/mL and Alexa 633 Phalloidin (Life Technology) overnight, washed four times with PBS for 15 min each, and mounted in VECTASHIELD mounting medium (Vector Laboratories).

Statistics.

Prism 6 (GraphPad Software) was used for statistical analyses and graphical presentations.