Heart- and Muscle-Derived Signaling System Dependent on MED13 and Wingless Controls Obesity in Drosophila

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

Ji-Hoon Lee, Rhonda Bassel-Duby, and Eric N. Olson1

Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9148

Contributed by Eric N. Olson, May 23, 2014 (sent for review March 27, 2014) Obesity develops in response to an imbalance of energy homeo- Furthermore, Drosophila serves as a model system to under- stasis and whole-body metabolism. Muscle plays a central role in stand systemic effects of interorgan cross-talk via secreted the control of energy homeostasis through consumption of energy molecules (3, 16). and signaling to adipose tissue. We reported previously that MED13, Here, we show that Drosophila muscle modulates obesity a subunit of the Mediator complex, acts in the heart to control obe- through the function of MED13 in the context of the Wingless sity in mice. To further explore the generality and mechanistic basis (Wg) signaling pathway. In Drosophila, MED13 and MED12 of this observation, we investigated the potential influence of are encoded by skuld (skd) and kohtalo (kto), respectively (17). MED13 expression in heart and muscle on the susceptibility of Muscle-specific knockdown of skd or kto increases fat body mass Drosophila to obesity. Here, we show that heart/muscle-specific and triglyceride accumulation in adult flies. We describe a genetic knockdown of MED13 or MED12, another Mediator subunit, screen for muscle-secreted obesity regulators, which revealed Wg increases susceptibility to obesity in adult flies. To identify possible as a muscle-derived suppressor of obesity. Similarly, inhibition of muscle-secreted obesity regulators, we performed an RNAi-based Armadillo (Arm), the Drosophila β-catenin ortholog and tran- genetic screen of 150 genes that encode secreted proteins and scriptional effector of Wg signaling, suppresses obesity. We also found that Wingless inhibition also caused obesity. Consistent identify functional relationships between skd and the Wg pathway with these findings, muscle-specific inhibition of Armadillo, the in which a skd-null mutation dominantly enhances the muscle downstream transcriptional effector of the Wingless pathway, phenotype resulting from arm knockdown, and wg acts down- also evoked an obese phenotype in flies. Epistasis experiments stream of skd in the regulation of fat accumulation. Our findings further demonstrated that Wingless functions downstream of indicate that Wg acts as an effector of MED13 function in muscle MED13 within a muscle-regulatory pathway. Together, these to suppress obesity in Drosophila. findings reveal an intertissue signaling system in which Wing- less acts as an effector of MED13 in heart and muscle and sug- Results gest that Wingless-mediated cross-talk between striated muscle Loss of MED13 Function in Muscle Increases Susceptibility to Obesity Drosophila and adipose tissue controls obesity in . This signaling in Drosophila. Based on our observation that cardiac deletion of system appears to represent an ancestral mechanism for the MED13 confers an obese phenotype and that cardiac-specific

control of systemic energy homeostasis. overexpression of MED13 prevents obesity in mice (4), we ex- CELL BIOLOGY amined whether muscle expression of MED13, encoded by skd, Skuld | kohtalo | myokines | metabolic syndrome regulates fat accumulation in Drosophila. We performed RNAi- mediated knockdown experiments using the UAS/Gal4 system besity is a systemic disorder caused by an energy imbalance and expressed UAS-RNAi targeting skd mRNA with the Mef2- Oin which energy input exceeds energy utilization, resulting Gal4 driver, which directs the expression of UAS constructs in in accumulation of excess body fat. Muscle plays a central role in somatic, cardiac, and visceral muscle tissues (18–21). By 3 wk systemic energy homeostasis by consuming nutrients and sig- of age, we observed increased abdominal fat bodies in adult – naling in an endocrine manner to other tissues (1 3). Thus, there Mef2>skd RNAi flies (Fig. 1A). Lipid droplets in the fat body has been intense interest in identifying secreted factors from cells were also enlarged as seen by Nile Red stain (Fig. 1B). muscle that modulate the function of adipose tissue. Consistently, total triglyceride amounts were significantly increased Previously, we reported that cardiac deletion of MED13, a subunit of the Mediator complex, increases susceptibility to Significance 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 Obesity is a major health epidemic and develops as a result of Mediator is a conserved multisubunit complex that mediates the imbalanced energy homeostasis. Previously, we reported that interaction between RNA polymerase II and transcription fac- cardiac expression of MED13, a subunit of the Mediator com- tors and therefore governs transcription in all eukaryotes (5). plex, controlled systemic energy homeostasis in mice such that MED13 and MED12 are among four subunits of the auxiliary increased or decreased expression of MED13 caused leanness or obesity, respectively. Here, we report that MED13 also acts kinase module, which confers additional regulatory functions Drosophila to the Mediator complex (6). Expression profiling of yeast within muscle of to control obesity. The secreted peptide Wingless acts as a downstream effector of MED13 to mutants or gene-depleted Drosophila cell lines revealed a close mediate cross-talk with adipose tissue and suppress obesity. correlation between the gene-expression programs controlled by Our work reveals a conserved signaling system in muscle in MED13 and MED12, suggesting their concerted actions in gene which MED13 and Wingless act as key controllers of obesity. regulation (7, 8). Drosophila provides a powerful model system for the genetic Author contributions: J.-H.L., R.B.-D., and E.N.O. designed research; J.-H.L. performed re- analysis of obesity (9–11). The processes that regulate energy search; J.-H.L., R.B.-D., and E.N.O. analyzed data; and J.-H.L., R.B.-D., and E.N.O. wrote homeostasis, such as energy storage and mobilization of fat in the paper. adipose tissue of the fat body, as well as the genetic pathways The authors declare no conflict of interest. governing such functions, are conserved in flies (12). Perturba- 1To whom correspondence should be addressed. E-mail: [email protected]. tions of such pathways in flies have been shown to generate This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. phenotypes relevant to human diseases, including obesity (13–15). 1073/pnas.1409427111/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1409427111 PNAS | July 1, 2014 | vol. 111 | no. 26 | 9491–9496 Downloaded by guest on September 25, 2021 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). 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 Fig. 1. MED13 expression in muscle regulates obesity. (A) Abdominal fat by extracellular factors secreted by muscle cells. To test this 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 hypothesis, we performed an RNAi-based genetic screen using Mef2>skd RNAi or Mef2>gfp (control) flies stained with Nile Red (red) and Mef2-Gal4 to identify muscle-secreted proteins that, when in- Phalloidin (green). (Scale bar: 50 μm.) (C–E) Relative triglyceride amounts of hibited, caused an obese phenotype, similar to what was ob- adult females with muscle-specific knockdown of skd, kto,orluciferase served with skd or kto knockdown in muscle. We analyzed 182 (control) using Mef2-Gal4 (C), Mhc-Gal4 (D), or Tin-Gal4 (E). Error bar, SEM; RNAi lines targeting 150 genes that encode secreted proteins. *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).

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). 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. 2. Flies with MED13 or MED12 knockdown display increased suscepti- (Fig. 1E). We also tested whether muscle-specific knockdown bility to obesity. (A) Age-dependent changes in total triglyceride levels. of kto, which encodes MED12, affected fat accumulation. Mef2-Gal4 was used to drive knockdown of skd or kto. Control 1, Mef2-Gal4 Indeed, flies with RNAi knockdown of kto in muscle or heart alone; control 2, Mef2>gfp.(B) Effects of high-fat diet on total triglyceride using Mef2-Gal4, Mhc-Gal4, or Tin-Gal4 displayed increased amounts in flies with the indicated genotypes. Twelve-day-old females total body triglyceride levels similar to the effect of skd 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 knockdown (Fig. 1 C E). > Resistance to starvation is a characteristic of obese flies (25). Mef2 kto RNAi increased triglycerides by 22% and 23% on average, respectively, but the differences were statistically insignificant (P > 0.05). With We tested whether skd or kto knockdown in muscle conferred high-fat food, Mef2>skd RNAi and Mef2>kto RNAi caused an increase of resistance to starvation. Three-week-old flies with Mef2-Gal4 triglycerides in flies to 57% and 33% on average, respectively. Control driven knockdown of skd or kto survived substantially longer harbors Mef2>gfp. Error bar, SEM; NS, not significant; *P < 0.05; **P < 0.01.

9492 | www.pnas.org/cgi/doi/10.1073/pnas.1409427111 Lee et al. Downloaded by guest on September 25, 2021 Relative total triglyceride levels of these flies at 4 wk of age were understand the nature of this lethality, the somatic muscle measured in comparison with the average of six controls (Table structure of the embryos was examined. Embryos expressing + + − + S1). Because skd knockdown using either Mef2-Gal4 or Mhc- luciferase RNAi in muscle in a skd / or skd / background Gal4 caused the fat accumulation phenotype (Fig. 1 C and D), maintained intact muscle patterns (Fig. 4 A and B). A portion + + we performed the knockdown screen again using the same set of of embryos expressing arm RNAiinmuscleinaskd / back- RNAi lines expressed with Mhc-Gal4 (Table S2). We identified ground had intact muscle patterns (Fig. 4C) whereas other six RNAi lines that increased total triglyceride levels greater than embryos with the same genotype displayed patterning defects 60% as a result of knockdown with both Mef2-Gal4 and Mhc- in the somatic musculature as exemplified by defects in lateral Gal4 (Table 1). transverse (LT) and dorsal muscles (Fig. 4C′). However, all observed embryos expressing arm RNAi using Mef2-Gal4 in − + Wg Signaling in Muscle Suppresses Obesity. We identified wg from the skd / background had patterning defects in their somatic the genetic screen described above (Table 1) and confirmed the musculature (Fig. 4 D and D′). effect of wg using two independent RNAi lines. Flies expressing either wg RNAi with Mef2-Gal4 showed increased abdominal fat The Epistatic Relationship of Wg and MED13 in Muscle for Obesity body mass (Fig. 3A). The lipid droplets therein were enlarged Control. Our finding that overexpression of wg in muscle caused (Fig. 3B), and total triglyceride amounts of the flies were also a lean phenotype (Fig. 3) whereas skd knockdown in muscle increased (Fig. 3C). To confirm the effect of wg knockdown in caused an obese phenotype (Fig. 1), as well as the functional muscle on total triglycerides and to test the heart-specific effect interaction between skd and arm (Fig. 4), raised the possibility of wg knockdown, we used Mhc-Gal4 and Tin-Gal4, respectively, that wg and skd act within a linear pathway in muscle to regulate to express wg RNAi and found that knockdown of wg with either obesity. To address this possibility, we tested the epistatic re- of the drivers resulted in a substantial increase of body tri- lationship between wg and skd. Flies expressing either skd RNAi glyceride content (Fig. 3 D and E). or wg cDNA or both with Mef2-Gal4 were grown for 4–4.5 wk, Consistent with the results from wg knockdown experiments, and their total triglyceride levels were compared. Total tri- wg overexpression in muscle decreased fat accumulation. Two glyceride levels of flies with both skd knockdown and wg over- independent UAS-wg cDNA lines were tested with Mef2-Gal4. expression were significantly decreased compared with those of These flies showed a decrease of abdominal fat body mass and flies with skd knockdown, but indistinguishable from those of lipid droplet size (Fig. 3 A and B). Total triglyceride levels in flies with wg overexpression alone (Fig. 5A). We also performed those flies were also decreased significantly (Fig. 3C). Reduced the same experiments using Mhc-Gal4 and Tin-Gal4 and obtained fat accumulation was also observed in flies overexpressing wg consistent results (Fig. 5 B and C). These data strongly support with Mhc-Gal4 or Tin-Gal4 (Fig. 3 D and E). the conclusion that muscle-secreted Wg acts as a downstream To investigate whether autonomous Wg signaling activity in effector of skd function in muscle to suppress fat deposition in muscle has a role in regulating obesity, we tested the effect on fat the fat body. accumulation of arm, encoding the transcriptional effector of Wg Finally, we tested the effect of Wg activation in the fat body. signaling, in muscle. The expression of arm RNAi using Mef2- When wg was overexpressed using the fat body-specific Dcg-Gal4

Gal4 resulted in increased abdominal fat bodies containing en- (29, 30), it caused lethality at the pupal stage and severe re- CELL BIOLOGY larged lipid droplets (Fig. 3 A and B). Total triglyceride levels duction of fat body mass in the abdominal region of third instar were also increased by muscle-specific knockdown of arm (Fig. larvae (Fig. 5D). These findings support a model in which 3C). In addition, using Mhc-Gal4 and Tin-Gal4 to knock down muscle-secreted Wg acts on the fat body to inhibit obesity (Fig. 6 arm, we observed an increase in total triglyceride levels (Fig. 3 D and Discussion). and E). Conversely, muscle-specific overexpression of arm either as a wild-type (armS2) or a constitutively active (armS10) form Discussion decreased abdominal fat body mass, lipid droplets in the fat Our results reveal a role of muscle in systemic regulation of body, and total triglyceride amounts (Fig. 3). obesity via the function of MED13 in Drosophila. We performed a genetic screen and identified muscle-secreted obesity- Functional Interaction Between skd and Arm in Muscle. To test the regulating factors, including Wg, and demonstrated that Wg functional interaction of the Wg pathway and MED13 in muscle, signaling in muscle is necessary and sufficient to suppress we performed genetic-interaction experiments between arm obesity. Furthermore, we showed that a skd-null mutation domi- and skd. Flies with arm knockdown using Mef2-Gal4 are viable nantly enhances the arm phenotype in muscle and that wg is without morphological defects. skdT606 is a null allele that is epistatic to skd, suggesting that Wg is a downstream effector of homozygous lethal (24), but skdT606 heterozygotes are viable MED13 in muscle. without morphological defects. However, arm knockdown using Our results reveal that MED13 in Drosophila muscle functions − + Mef2-Gal4 in a skdT606 heterozygous (skd / ) background to suppress obesity based on several criteria, such as histology, caused complete lethality, which was fully penetrant. To better measurement of whole-body triglycerides, tolerance to starvation

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.

Lee et al. PNAS | July 1, 2014 | vol. 111 | no. 26 | 9493 Downloaded by guest on September 25, 2021 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, armS10 cDNA (constitutively active), armS2 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.

stress, and susceptibility to high-fat diet. Similarly, muscle-specific muscle regulates fat accumulation in Drosophila is consistent knockdown of MED12 also increased fat accumulation, suggesting with our earlier observation with mice (4) and suggests that the that MED12 and MED13 function similarly in the control of fat function of cardiac MED13 in systemic regulation of fat storage deposition in Drosophila. Our finding that MED12 and MED13 represents an ancestral mechanism conserved in metazoans. modulate energy homeostasis adds to a growing number of Although it seems most likely that the effect of MED13 on examples in which components of the kinase module of the obesity is mediated by overall changes in metabolism, it is also Mediator complex influence metabolic signaling on an organ- conceivable that changes in feeding behavior contribute to the ismal level. For example, the other two components of the kinase obesity phenotypes we observed. module, Cyclin-dependent kinase 8 and Cyclin C, have also been Knockdown of MED12 and MED13 using drivers that are reported as negative regulators of fat accumulation in flies and active specifically in the heart using Tin-Gal4 or generally in mice (31). Our finding that the activity of MED13 in cardiac all muscles using Mef2-Gal4 or Mhc-Gal4 commonly evoked

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 skdT606. 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.

9494 | www.pnas.org/cgi/doi/10.1073/pnas.1409427111 Lee et al. Downloaded by guest on September 25, 2021 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). Further- more, 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 reg- Fig. 5. Wg functions downstream of MED13 in muscle to regulate obesity. ulation, one function of muscle-secreted Wg might be to act (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 on muscle. Accordingly, the nonautonomous function of Wg to wg cDNA or skd RNAi or both in muscle using Mef2-Gal4 (A), Mhc-Gal4 (B), suppress obesity may occur through autonomous Wg signal ac- or Tin-Gal4 (C). Error bar, SEM; NS, not significant; **P < 0.01; ***P < 0.001. tivity in muscle. However, if MED13 functions at the level of (D) Images of third instar larvae with Wg overexpression using fat body- transcriptional control of Wg target genes and the sole function specific Dcg-Gal4. Control was Dcg-Gal4 alone. Arrows indicate abdominal of muscle-secreted Wg ligand is to activate the Wg signal “in” region where fat bodies are severely reduced. 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 comparable obesity phenotypes. Thus, we conclude that MED12 than muscle for its nonautonomous effect. If so, which tissue and MED13 can control metabolic signaling from the heart, may be the target? Ectopic expression of Wg using a fat body- consistent with our prior conclusions regarding the functions specific Dcg-Gal4 decreased larval abdominal fat body mass, of MED13 in the mouse heart. However, these Gal4 drivers do which demonstrates the role of Wg signaling in the fat body for not enable us to reach conclusions regarding the specific role fat-mass regulation. Similarly, in mammals, autonomous activation of somatic or visceral muscle in this signaling process because of the Wnt pathway in adipose tissue decreases fat mass. Wnt sig- Mhc-Gal4 and Mef2-Gal4 are active in diverse muscle-cell types. naling blocks mammalian adipogenesis in vitro (42), and, in mice, Given that MED12 and MED13 are ubiquitously expressed, it activation of the canonical Wnt pathway in adipocytes by ectopic is possible that they also act in nonmuscle tissues to regulate expression of Wnt10b, a Wnt ligand, inhibits obesity (43, 44). Fur- metabolic homeostasis. thermore, autonomous activation of the Wnt pathway in adipose We hypothesized that muscle-secreted factors mediate the progenitors with constitutively active β-catenin expression decreases function of MED13 in Drosophila muscle to suppress systemic fat fat mass (45). Therefore, the reduced fat mass in Dcg > wg larvae accumulation. To identify such factors, we screened for muscle- indicates that autonomous Wg signaling activity in the fat body secreted obesity-regulating proteins using two different muscle serves as a regulator of fat mass. Considered together with our data drivers, Mef2-Gal4 and Mhc-Gal4. We identified six genes that CELL BIOLOGY > showing that muscle-secreted Wg contributes to nonautonomous increased fat accumulation of flies in both screens by 60%, regulation of adiposity in vivo, we conclude that muscle serves as including the genes encoding (i) an antimicrobial peptide, Dip- a source of Wg to regulate adiposity by modulating Wg signaling tericin B (32); (ii)aDrosophila homolog of Angiotensin con- activity in fat body. However, we cannot rule out the possibility that verting enzyme (33); (iii) a G protein-coupled receptor ligand the systemic effect of Wg from muscle is mediated through an al- iv Drosophila SIFamide (34); ( ) one of seven Insulin/IGF homo- ternative tissue, such as nervous system (46). logs, Insulin-like peptide 4 (35); (v) a JAK/STAT signaling li- Wg acts on short- and long-range targets. Wg is highly hy- vi gand, Unpaired 3 (36); and ( ) Wg. Interestingly, it has been drophobic and has been shown to diffuse through the extracel- shown recently that MED13 and MED12 are required for the lular space and act on long-range targets by associating with expression of Diptericin B in response to Immune Deficiency solubilizing molecules such as lipoprotein particles and Secreted (IMD) pathway activation (8), suggestive of additional regula- Wg-interacting molecule (47, 48). Furthermore, Wnt-1 has been tory functions of MED13 and the genes identified from our identified in serum, and decreased Wnt-1 levels in serum cor- screens beyond obesity control. relate with premature myocardial infarction and metabolic syn- We demonstrated that Wg and its autonomous signaling ac- drome (49), suggesting that Wg may act on remote organs as an tivity, controlled by Arm, in muscle are necessary and sufficient endocrine factor. Therefore, we propose a model in which for systemic regulation of obesity in vivo. Previously, the corre- muscle-secreted Wg is a downstream effector of MED13 and lation between obesity and the expression of genes involved in acts both to activate the signal in muscle and to act on the fat the Wnt signaling pathway in heart has been raised from tran- body ultimately to achieve systemic inhibition of obesity. scriptome 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 Fig. 6. A model of muscle-derived signaling via MED13 and Wg for obesity mesoderm and the embryonic musculature (39). Our findings re- control. Expression of MED13 or Wg in muscle suppresses fat accumulation veal a close functional connection between MED13 and Arm, in the fat body, and Wg acts downstream of MED13. Muscle-secreted Wg suggestive of the role of MED13 in Wg target gene expression. In activates the Wg signal in both muscle and fat body.

Lee et al. PNAS | July 1, 2014 | vol. 111 | no. 26 | 9495 Downloaded by guest on September 25, 2021 Materials and Methods Technology) overnight, washed four times with PBS for 15 min each, and Fly Stocks and Genetics. Fly stocks used are described in SI Materials and mounted in VECTASHIELD mounting medium (Vector Laboratories). Methods. Flies were maintained in vials with Cornmeal-Molasses-Yeast medium except for experiments using high-fat food, which was 30% (wt/vol) Statistics. Prism 6 (GraphPad Software) was used for statistical analyses and coconut oil (MP Biomedicals) added to normal food, as described (28). De- graphical presentations. tailed procedures of fly genetics are available in SI Materials and Methods. ACKNOWLEDGMENTS. We thank Jonathan Graff [University of Texas South- Triglyceride Assay. Triglyceride assay was performed as described with mod- western Medical Center (UT Southwestern)], Michael Buszczak (UT South- western), Bum-Kyu Lee (UT Austin), Aaron Johnson (University of Colorado ifications (30) and is available in SI Materials and Methods. Denver), Jin Seo (Rogers State University), and members of the E.N.O. laboratory for helpful discussion; Dylan Tennison and Evelyn Tennison for Immunohistochemistry. Antibodies used were anti-Mhc (a gift of Bruce Paterson, technical support; Jose Cabrera for graphics; Jessica Treisman (New York National Cancer Institute, Bethesda) and anti-GFP (Torrey Pines Laboratories). University), Bruce Paterson (National Cancer Institute), Jonathan Graff (Uni- Embryos were immunostained as described (50). versity of Texas Southwestern Medical Center), and Janice Fischer (University of Texas at Austin) for providing reagents; and the Transgenic RNAi Project at Harvard Medical School [National Institutes of Health (NIH)/National In- Nile Red Staining. To visualize adult abdominal fat bodies, a vertical incision stitute of General Medical Sciences Grant R01-GM084947] for providing was made along the ventral abdomen of female flies. Other internal organs transgenic RNAi fly stocks. This work was supported by NIH Grants HL- were removed and fixed in 4% paraformaldehyde for 15 min. Images were 077439, HL-111665, HL-093039, DK-099653, and U01-HL-100401, grants from taken using a Zeiss AxioCam. Fixed abdomens were stained with Nile Red the Cancer Prevention and Research Institute of Texas, and Robert A. Welch (Fluka) at a concentration of 10 μg/mL and Alexa 633 Phalloidin (Life Foundation Grant 1-0025 (to E.N.O.).

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