Reversal of hyperactive Wnt signaling-dependent PNAS PLUS defects by peptide boronic acids

Tianyi Zhanga,1, Fu-Ning Hsub,1, Xiao-Jun Xieb,1, Xiao Lib, Mengmeng Liub, Xinsheng Gaob, Xun Peia, Yang Liaoa, Wei Dua,2, and Jun-Yuan Jib,2

aBen May Department for Cancer Research, The University of Chicago, Chicago, IL 60637; and bDepartment of Molecular and Cellular Medicine, College of Medicine, Texas A&M University Health Science Center, College Station, TX 77843

Edited by Norbert Perrimon, Harvard Medical School, Boston, MA, and approved July 17, 2017 (received for review December 22, 2016) Deregulated Wnt signaling and altered lipid metabolism have been the Wnt/β-catenin pathway inhibits adipocyte differentiation and linked to obesity, diabetes, and various cancers, highlighting the development (16–19); for example, activation of Wnt signaling in importance of identifying inhibitors that can modulate Wnt signal- cultured mouse preadipocyte 3T3-L1 cells impaired adipogenesis, ing and aberrant lipid metabolism. We have established a Drosophila whereas inhibition of β-catenin activity promoted this process (20). model with hyperactivated Wnt signaling caused by partial loss of In addition, in vivo studies have shown that overexpression of axin, a key component of the Wnt cascade. The Axin mutant larvae Wnt10b or β-catenin in adipose tissue impairs the formation of are transparent and have severe adipocyte defects caused by up- white and brown adipose tissue and causes fibrosis in mice (19, regulation of β-catenin transcriptional activities. We demonstrate 21). Conversely, the loss of β-catenin activity in mouse embryonic pharmacologic mitigation of these phenotypes in Axin mutants by mesenchyme switches the fate of normal uterine smooth muscle to identifying bortezomib and additional peptide boronic acids. We in vivo (22), and mutations in the human WNT10B show that the suppressive effect of peptide boronic acids on hyper- are associated with early onset of obesity (23). Although active Wnt signaling is dependent on α-catenin; the rescue effect is these findings support the idea that Wnt signaling inhibits adi- completely abolished with the depletion of α-catenin in adipocytes. pogenesis, less is known about the roles of the canonical Wnt These results indicate that rather than targeting the canonical Wnt pathway in regulating different aspects of fat metabolism, in- signaling pathway directly, pharmacologic modulation of β-catenin cluding lipogenesis, lipolysis, and fatty acid β-oxidation. This may activity through α-catenin is a potentially attractive approach to be due to the intertwined nature of these metabolic and cellular CELL BIOLOGY attenuating Wnt signaling in vivo. processes in mammalian adipocytes (14, 24). Drosophila represents a powerful model system for studying axin | catenin | adipocyte | peptide boronic acid | Drosophila mechanisms that control lipid metabolism (25–27), for several reasons. First, there is high conservation in the enzymes, regula- he controls many fundamental aspects tory factors, pathways, tissues, and organs that regulate lipogenesis Tof normal development and tissue homeostasis in metazoans and adipogenesis between Drosophila and mammals. For example, by regulating cell differentiation, proliferation, migration, and the lipogenic enzymes found in mammals, including fatty acid metabolism (1–4). A key downstream effector of the canonical Wnt synthase, acetyl-CoA carboxylase, and Acyl-CoA synthetase, have signaling pathway is β-catenin, which functions as a cofactor for the virtually the same biochemical activities as the homologous en- T-cell factor and lymphoid-enhancing factor (TCF/LEF) family of zymes in Drosophila. At the tissue level, mature adipocytes in transcription factors in nucleus. In addition, β-catenin also mediates Drosophila fat bodies contain numerous lipid droplets and store cell–cell adhesion by serving as a component of adherens junctions most of the TG, mimicking the role of mammalian adipose tissues. at the cell membrane. β-catenin links adherens junctions and cy- toskeleton by binding to the transmembrane cadherin, as Significance wellasmicrofilamentsthrough the adaptor protein α-catenin (α-Cat) (5–7). Membrane-associated β-catenin is stable and may Deregulated Wnt signaling is often observed in diverse human represent the major subcellular pool of β-catenin. In contrast, the diseases, including cancers, and is a potential therapeutic target. soluble cytoplasmic pool of β-catenin is actively degraded by the Here we report that hyperactivated Wnt/Wg signaling disrupts proteasome in the absence of a Wnt signal. This is accomplished by fat metabolism in Drosophila larvae, and that peptide boronic the β-catenin destruction complex, which includes adenomatous acids, a unique class of proteasome inhibitors, can potently polyposis coli (APC), the scaffolding protein Axin (Axn), the Ser/ rescue the fat defects by inhibiting Wg signaling through sta- Thr kinase glycogen synthase kinase 3 (GSK3), and its priming bilization of α-catenin. We show that Axn127 mutant is an at- kinase casein kinase 1 (CK1) (8–10). The destruction complex is tractive system for screening for and optimizing small molecules inhibited on Wnt stimulation, allowing β-catenin to accumulate and that target Wnt signaling and proteasome in vivo. This work translocate into the nucleus, thereby activating expression of the suggests that pharmacologic strategies for stabilizing α-catenin Wnt target (4). Mutations of certain components of the Wnt may represent an attractive approach to attenuate Wnt sig- signaling pathway have been implicated in abnormal development naling, rather than directly targeting components of the Wnt and various diseases, including diabetes and a variety of human signaling pathway. cancers, particularly colorectal cancer (1–4, 11). Despite consider- able efforts, the development of inhibitory compounds that can Author contributions: T.Z., F.-N.H., X.-J.X., W.D., and J.-Y.J. designed research; T.Z., F.-N.H., target the Wnt signaling pathway for therapeutics remains an at- X.-J.X., X.L., M.L., X.G., Y.L., and J.-Y.J. performed research; X.P. contributed new re- tractive but elusive goal (12, 13). To our knowledge, there is no agents/analytic tools; T.Z., F.-N.H., X.-J.X., X.L., W.D., and J.-Y.J. analyzed data; and T.Z., F.-N.H., X.-J.X., W.D., and J.-Y.J. wrote the paper. currently Food and Drug Administration (FDA)-approved drug to treat diseases by inhibiting Wnt signaling activities. The authors declare no conflict of interest. Adipocytes play critical roles in maintenance of fat and energy This article is a PNAS Direct Submission. 1 homeostasis in animals. Adipogenesis refers to the cellular pro- T.Z., F.-N.H., and X.-J.X. contributed equally to this work. 2 cess of adipocyte differentiation, whereas lipogenesis is defined To whom correspondence may be addressed. Email: [email protected] or ji@medicine. tamhsc.edu. as fatty acid and triglyceride (TG) biosynthesis from acetyl-CoA This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. (14, 15). Multiple lines of evidence have shown that activation of 1073/pnas.1621048114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1621048114 PNAS | Published online August 21, 2017 | E7469–E7478 Downloaded by guest on September 23, 2021 Similarly, the function and regulation of the key regulators, such To analyze the defective fat metabolism at the cellular level, we as the and Wnt signaling pathways, are highly conserved stained the abdominal adipocytes in WT and Axn127 mutant larvae but simpler in Drosophila than in mammals. Second, Drosophila using boron-dipyrromethene (BODIPY). The control adipocytes provides a plethora of sophisticated genetic tools and molecular were largely uniform in size, with numerous oil droplets accu- markers for the functional analysis of adipocyte biology, which mulated within each cell (Fig. 1G and Fig. S2A); however, many of 127 facilitates analyses that are difficult to perform in other experi- the adipocytes in the Axn mutants were smaller and with fewer mental systems. Third, recent studies have identified Drosophila as oil droplets compared with WT (yellow arrow in Fig. 1H and Fig. an excellent model for screening small molecule compounds and S2C), mixed with occasional large adipocytes (red arrow in Fig. subsequent mechanistic studies (28–31). 1H). The strikingly reduced number of oil droplets and size of 127 Here we report our characterization of Axn mutants as a adipocytes in Axn mutants are consistent with the reduced TG unique Drosophila model that propagates a hyperactive Wnt levels of whole larvae as described above. signaling pathway, leading to dysregulated fat metabolism. This Defective Fat Accumulation in Axn127 Mutants Is Caused by a Gain in adipocyte defect was potently restored by three peptide boronic 127 acids, and the underlying mechanism was found to be mediated Wg Signaling. Exon 11 of the Axn allele is replaced by a re- petitive heterochromatin sequence, resulting in a mutant Axn by α-Cat. protein that lacks part of the DIX domain in the C terminus (32). Results The DIX domain is required for homopolymerization and het- Axn127 127 eropolymerization of Axn and (Dsh, or Dvl in Mutant Larvae Are Defective in Fat Metabolism. The Axn – homozygous mutants (genotype: w1118; +;FRT82BAxn127)survive mammals) (10, 33 35). We also observed significantly lower levels of Axn protein in Axn127 mutant larvae relative to control larval stages and mostly die at the pupal stage (32). Interestingly, (Fig. S3A), suggesting that both the damaged DIX domain and compared with wild-type (WT) larvae (w1118;Fig.1A), Axn127 the decreased protein level may contribute to the loss of function homozygous mutants at the third-instar wandering stage are more in Axn127 phenotypes. transparent in the abdominal region (Fig. 1B). To test whether Consistent with the idea that Axn is a negative regulator of these mutant larvae have less fat accumulation in the abdominal Armadillo (Arm, the Drosophila ortholog of β-catenin) (36), the fat body, the adipocyte tissue that predominantly controls lipid level of Arm protein is significantly increased in Axn127 mutant homeostasis in Drosophila, we quantified the fat levels using Oil larvae (32). Thus, we investigated whether Arm activity, and hence Red O to stain the neutral fat and a TG assay kit to directly Wg signaling, is increased by analyzing the expression of Arm- measure TG levels in the third instar wandering larvae. Compared – 127 activated genes, such as naked cuticle (nkd)andNotum (37 39). with the WT control, Axn mutant larvae showed significantly The mRNA levels of these genes were indeed significantly in- lower Oil Red O staining (Fig. 1C) and TG levels (Fig. 1D). creased in Axn127 mutant larvae, whereas the mRNA levels of arm We next quantified the levels of different types of diacylglycerol was not altered and Axn mRNA was significantly reduced in the (DG), free fatty acid (FFA), phosphatidylglycerol (PG), and TG in 127 127 Axn mutants (Fig. S3B). These findings suggest that the en- WT and Axn mutant larvae using mass spectrometry. This hanced Arm target gene expression in Axn127 homozygous mu- analysis revealed a significant increase in 9 of 11 types of FFA tants is correlated with increased Wg signaling activity. (Fig. 1E and Fig. S1A), but a striking reduction in 24 of 26 types of If gain of Wg signaling underpins the defective fat metabolism 127 TG (Fig. 1F and Fig. S1B)intheAxn mutants compared with and aberrant adipocytes, then genetically down-regulation of Wg the WT controls. The FFAs and TGs that did not follow this trend signaling in Axn127 mutants should rescue these defects. Indeed, were also very small components of WT and Axn127 mutant larvae knockdown of Arm by RNAi in the Axn127 homozygous back- (Fig. S1 A and B). Significant reductions in several DGs (Fig. S1C) ground significantly suppressed the fat defects (Fig. 2A). The key and PGs (Fig. S1D)werealsoobservedintheAxn127 mutants. factors downstream of the Wg signaling cascade include These results suggest that the Axn127 mutants may have impaired encoded by pangolin (pan; an ortholog of transcription factor lipid metabolism. TCF), as well as two transcriptional cofactors for Pan encoded by

Fig. 1. The Axn127 mutant larvae are defective in fat metabolism. (A and B) Representative images of the control (A; w1118) and the Axn127 homozygous mutants (B) at the L3 wandering stage under a dis- secting microscope. (C) Quantification of the Oil Red O staining (n = 14). Results are normalized to the control (w1118). (D) Quantification of TG levels (n = 3 biological repeats). Results are normalized to the control (w1118). (E and F) Mass spectrometry analysis of several representative types of FFAs and TGs in Axn127 mutant larvae (magenta) and control (w1118, black). Detailed results of different types of lipids are shown in Fig. S1.(G and H) Confocal images of fat body stained with DAPI, Phall, and BODIPY to show nuclei, cell boundary, and lipid droplets, respectively. ***P < 0.001.

E7470 | www.pnas.org/cgi/doi/10.1073/pnas.1621048114 Zhang et al. Downloaded by guest on September 23, 2021 PNAS PLUS CELL BIOLOGY

Fig. 2. Defective fat metabolism in Axn127 mutants is caused by gain of the Wg signaling and occurs during L3 stages. (A–F) Images of LL3 larvae of the indicated genotypes under a dissecting microscope. (Scale bar in A:1.0mm.)Genotypes:(A) arm-Gal4/+;Axn127/ UAS-armRNAi, Axn127;(B) arm-Gal4/+;Axn127/UAS-panRNAi, Axn127;(C) arm-Gal4/UAS-lgsRNAi;Axn127;(D) arm-Gal4/UAS-pygoRNAi;Axn127;(E) SREBP-Gal4/UAS-lgsRNAi;Axn127;(F) SREBP-Gal4/+;Axn127/UAS-panRNAi,Axn127. (G and H) Representative fluorescent images of fat body stained with Phall (red) and DAPI (blue) of larvae during EL3 stage (G and H), ML3 stage (G′ and H′), and LL3 stage (G′′ and H′′). Arrows in H′ and H′′ point to adipocytes with different sizes: yellow arrows indicate small adipocytes, and red arrows indicate big adipocytes.

legless (lgs)andpygopus (pygo) (4, 40, 41). Similar to the depletion 2H′). This heterogeneic adipocyte size in Axn127 mutants was more of Arm, knockdown of pan (Fig. 2B), lgs (Fig. 2C), or pygo (Fig. evident at the LL3 stage (arrows in Fig. 2H′′). These observations 2D) strongly suppressed the fat body defects in Axn127 mutants. In show that the size of the majority adipocytes in the abdominal fat the case of lgsRNAi, TG levels were restored to those of controls body failed to increase in Axn127 mutants during ML3 and (Fig. S3C). LL3 larval stages, indicating that the mechanism controlling fat Given the ubiquitous expression of arm-Gal4, we tested whether homeostasis in adipocytes is defective in Axn127 mutants. depletion of these factors using Gal4 lines with more restricted expression patterns could have similar effects. For this experiment, Defects in Both Fat Body and Gut Contribute to Aberrant Fat 127 we used SREBP-Gal4 line, which is expressed predominantly in Metabolism in Axn Mutants. Axn127 is an allele induced by ran- the fat body and part of the intestine (42). We found that dom mutagenesis (32). To rule out the possibility that the fat body knockdown of either lgs (Fig. 2E)orpan (Fig. 2F)bySREBP- defects in Axn127 are caused by genetic background, we combined – 127 EY10228 Gal4 driven RNAi also strongly suppressed the fat body defects in Axn over another allele, Axn , which is caused by a Axn127 mutants. Depletion of these components of the Wg sig- 127 transposon insertion near the transcription initiation site and naling pathway also increased the levels of TGs in Axn mutants represents a strong hypomorphic allele (Fig. S4A)(32,44).The (Fig. S3C). Taken together, these results suggest that adipocyte 127 EY10228 127 Axn /Axn transheterozygous mutants are transparent at defects in Axn mutant larvae are caused by defective degrada- the LL3 wandering stage (Fig. S4C, compared with the control in tion of Arm, thereby increasing Wg activity. 127 Fig. S4B), similar to the Axn homozygous mutants (Fig. 1B). 127 EY10228 Defects of Adipose Tissue in Axn127 Mutants Occur in L3 Stage. Adi- The Axn /Axn mutants also have significantly reduced TG levels and adipocyte size (Fig. S4 J and L). Importantly, the pocytes that undergo endoreduplication increase in size by accu- EY10228 mulating oil droplets during the larval stage (43). To identify the Axn allele is unique in that the transposon contains the 127 “ ” earliest stage for adipocyte defects in Axn mutants, we analyzed upstream activating sequence (UAS) in the plus orientation, the morphology of adipocytes in early-stage L3 (EL3), mid-stage which allowed us to express WT Axn when combined with dif- L3 (ML3), and late-stage L3 (LL3) larvae. Adipocytes in the ferent Gal4 drivers (Fig. S4A). Indeed, ubiquitous expression of Axn127 mutant larvae in EL3 (Fig. 2H) were of uniform size, similar WT Axn strongly rescued defective fat accumulation (Fig. S4D), to the WT control cells at the same stage (Fig. 2G); however, from suggesting that this approach can be used to examine potential EL3 to ML3, adipocytes in controls increased in size (Fig. 2G′), tissue-specific contributions to the defective fat metabolism in the whereas only a few adipocytes in Axn127 mutantsgrewlarger(Fig. Axn127 mutants.

Zhang et al. PNAS | Published online August 21, 2017 | E7471 Downloaded by guest on September 23, 2021 To test whether restoring Axn in fat bodies alone can rescue the there has been considerable interest in developing small mole- defects in fat metabolism, we used a larval and adult fat body- cules that can modulate Wnt signaling. However, this effort has specific FB-Gal4 line (45, 46), and observed partial rescue (Fig. S4 been hampered by the lack of an efficient, robust, and cost- E and J). Similarly, restoring Axn in the midgut and hindgut using effective experimental system for evaluating and screening Gal4 lines specifically expressed in these tissues (47) partially compounds that target Wnt signaling in vivo. The fat body de- rescued the fat defects (Fig. S4 F and J). However, restoring Axn fects can be readily inspected under a microscope, are sensitive in the fat bodies, midgut, and hindgut together almost completely to genetic modulations by the Wg pathway components, and are rescued the defects of fat accumulation (Fig. S4 G and J). To mechanistically caused by hyperactive Wg signaling. Therefore, further test this effect, we used the SREBP-Gal4 line, which ex- we explored whether this system can be used to screen for small presses Gal4 in the fat bodies and part of the gut (42). As shown in molecules that can modulate the Wg signaling, which is highly Fig. S4 H and J, Axn expression driven by SREBP-Gal4 also conserved in evolution. strongly rescued the fat body defects in the Axn127/AxnEY10228 We first tested seven available compounds that have been mutants. Taken together, these observations suggest that defective reported to target various components of the Wnt signaling functions of Axn in both the fat bodies and the gut contribute to pathway in vertebrates (52–56). Axn127 mutant larvae were fed the fat accumulation defects in the Axn127 mutants. with food mixed with each drug at a final concentration We next tested whether depletion of Axn by RNAi in fat bodies of ≥2.0 μM; this approach has been used previously to examine can mimic the effect of Axn127 mutation. We observed that de- the effects of drugs on Drosophila (28, 57). None of the seven pletion of Axn using FB-Gal4 generated fat bodies with occasional compounds showed any effect on modulation of fat body defects smaller adipocytes (yellow arrows in Fig. S5B′), whereas more in the Axn127 mutants (Table S1). In addition, we tested three smaller adipocytes were seen when Axn was depleted in an small molecule inhibitors (iCRT3, iCRT5, and iCRT14) known Axn127/+ background (Fig. S5C′), suggesting that Axn level is to block β-catenin–responsive transcription in cultured Dro- critical for the observed fat body defects. Interestingly, much sophila and mammalian cells (58). We did not observe any effects stronger effects were observed when similar experiments were on fat body defects when the Axn127 mutant larvae were fed with performed using the SREBP-Gal4 driver, which is expressed in these inhibitors at concentrations ranging from 1.0 to 20 μM both fat bodies and the gut. The larvae were transparent (Fig. S5E (Fig. S7). vs. the control in Fig. S5D), and most of the adipocytes were We next screened a library of 775 FDA-approved drugs to smaller and had fewer oil droplets (Fig. S5E′). These findings identify those that can rescue the fat body defects in the Axn127 show that depletion of Axn by RNAi can mimic the Axn127 adi- mutants (details in Materials and Methods), and identified borte- pocyte defects at the cellular level. zomib (BTZ, or PS-341; trade name Velcade), the first protea- We next asked whether inducing strong Wg signaling in fat some inhibitor approved to treat multiple myeloma and mantle bodies alone could lead to the defects in adipocytes and fat ac- cell lymphoma (59). As shown in Fig. 3A, feeding the Axn127 cumulation. To explore this, we tested whether expression of a mutants food supplemented with 2 μM BTZ strongly suppressed dominant negative form of Xenopus GSK3β (UAS-GSK3βDN), the fat body defects compared with the mutant larvae fed with the which is enzymatically inactive owing to mutation of a conserved food mixed with DMSO. We used a TLC assay to determine the lysine residue in the ATP-binding site (GSK3β-K85R) (48, 49), in levels of TG, DG, MG, and FFA, and found significantly greater fat bodies could mimic the phenotypes observed in Axn127 mu- reductions in TG levels and higher FFA levels in the Axn127 larvae tants. In a previous study, we showed that overexpression of (Fig. 3B, lane 2) compared with the WT control larvae (Fig. 3B, GSK3βDN induced strong defects in Drosophila eye development lane 1). These results are consistent with our lipidomic analysis (32). Remarkably, ectopic expression of GSK3βDN in the fat (Fig. 1). In support of our microscopic observations, feeding the bodies, driven by FB-Gal4, resulted in transparent larvae (Fig. Axn127 mutants with BTZ strongly increased TG levels and re- S4I), similar to the Axn127 (Fig. 1B)andAxn127/AxnEY10228 mutants duced FFA levels (Fig. 3B, compare lanes 4 and 2), suggesting that (Fig. S4C). At the cellular level, we observed heterogeneous adi- BTZ can rescue the fat defects in the Axn127 mutant larvae. pocytes (Fig. S4M) resembling the Axn127 and Axn127/AxnEY10228 To examine the effect of BTZ at the cellular level, we stained mutant adipocytes (Fig. 1H and Fig. S4L). Quantification of TG the adipocytes from BTZ-fed Axn127 mutant larvae with Nile levels confirmed a significant reduction in fat accumulation when Red. Compared with the DMSO-fed Axn127 controls that showed GSK3βDN was ectopically expressed in adipocytes (Fig. S4J). heterogeneity in adipocyte size and fat accumulation (Fig. 3D), These observations suggest that hyperactivated Wg activity in- the adipocytes from BTZ-fed Axn127 mutants displayed greater duced by overexpression of GSK3βDN in fat bodies alone is suf- cellular uniformity in size and fat storage (Fig. 3D′). In addition, ficient to induce the adipocyte defects similar to those observed in we identified 2 μM BTZ as the optimal dose; lower doses (1 μM; Axn127 mutants. Fig. S8) led to weak effects, whereas high concentrations (e.g., To further test whether increased Wg activity can have cell- 8.0 or 10 μM) were lethal to the larvae. Interestingly, 2 μM BTZ autonomous effects in adipocytes, we generated mutant clones of had a weak but opposite effect on fat accumulation in WT larvae Axn127 and the AxnS044230 allele (homozygotes of this stronger al- (Fig. 3 A and B), with a slight decrease in the size of the adi- lele are early larval-lethal) using an hs-Flp and trans-FRT ap- pocytes (Fig. 3C′, compared with the control shown in Fig. 3C). proach. Heat shock during embryogenesis led to rare clones in the Nevertheless, these observations show that BTZ can potently fat bodies (Fig. S6). We found that Axn127 (Fig. S6A)and suppress the fat body defects in the Axn127 mutant larvae. AxnS044230 (Fig. S6B) mutant cells were smaller than neighboring To further validate these observations, we investigated whether WT cells, with significantly reduced Nile Red staining. These second-generation proteasome inhibitors could have a similar findings suggest that loss of Axn in adipocytes can cause the rescue potential as BTZ for the fat defects in the Axn127 mutants. aforementioned adipocyte defects. Taken together, these obser- We tested five commercially available proteasome inhibitors: vations suggest that the adipocyte defects in Axn127 mutant larvae carfilzomib (CFZ; Kyprolis or PR-171), delanzomib (DLZ, or are caused by defects in both fat bodies and the gut, and that a gain CEP-18770), ixazomib (IXZ, or MLN-9708; trade name Ninlaro), in Wg activity in adipocytes is sufficient to affect lipid metabolism. marizomib (MRZ; NPI-0052 or salinosporamide A), and oprozo- mib (OPZ; ONX-0912 or PR-047) (59). Although all these com- Small Molecule Screens Using the Axn127 Fat Defect Phenotypes pounds can potently and specifically inhibit the 20S catalytic core Identify Peptide Boronic Acids. Because dysregulated Wnt activi- of proteasome, they have different structures and differ in their ties cause abnormal development and cancer in diverse tissues, reversibility of inhibition. DLZ, IXZ, and BTZ are peptide boronic as well as metabolic diseases in liver and adipocytes (50, 51), acid derivatives and are reversible proteasome inhibitors; CFZ and

E7472 | www.pnas.org/cgi/doi/10.1073/pnas.1621048114 Zhang et al. Downloaded by guest on September 23, 2021 BTZ Inhibits Expression of Genes Activated by the Wg Signaling PNAS PLUS Pathway. Fig. 2 and Fig. S3 show that the fat defects in Axn127 mutants are correlated with elevated expression of Wg target genes; thus, we investigated whether BTZ can affect the ex- pression of these Wg targets. We first analyzed the effect of BTZ on the expression of a reporter line nkd-lacZ (the nkd04869a al- lele), a lacZ enhancer trap line in the nkd gene (68). The nkd- lacZ reporter is not expressed in the fat bodies of WT or Axn127 larvae. In control larvae, the reporter is expressed at the boundary between the anterior hindgut and posterior midgut (Fig. 4A). In contrast, Axn127 mutant larvae have stronger ex- pression of nkd-lacZ, with signal expansion to the middle part of the midgut (Fig. 4B). When fed with food containing 2 μM BTZ, both the WT and Axn127 mutants exhibited reduced expression of nkd-lacZ, with a more apparent effect in the Axn127 mutants (Fig. 4 A′ and B′). In contrast, feeding these larvae with iCRT3 or iCRT14 had no effect on nkd-lacZ expression (Fig. S11). We next analyzed additional Wg target genes using quantita- tive RT-PCR of whole larvae fed with BTZ. The expression of Arm target genes Notum and nkd in BTZ-fed Axn127 mutant larvae were significantly lower than the expression levels mea- sured in control Axn127 mutants fed with DMSO, whereas no difference was found in the mRNA level of arm in BTZ-treated larvae (Fig. 4C). These observations suggest that BTZ can cor- rect the ectopically gained activity of the Wg signaling pathway in the gut of Axn127 mutants, consistent with the strong suppressive effects of BTZ on Axn127 fat defects (Fig. 3). We then explored the mechanisms underlying the suppressive CELL BIOLOGY effects of BTZ on the Wg signaling pathway. It is well established that β-catenin is degraded by the ubiquitin-proteasome pathway and that the stability of β-catenin is positively correlated with its activities (8, 69). Thus, based on the proteasomal inhibition of BTZ, we would expect to find that BTZ stabilizes β-catenin, thereby ectopically activating Wg signaling in WT larvae. Indeed, an increased Arm level was observed in WT larvae treated with BTZ (Fig. 4D). However, our results show that proteasome in- 127 Fig. 3. Peptide boronic acids rescue the fat defects in Axn127 mutants. hibitor BTZ also can down-regulate Wg activity in Axn mutants (A) Representative images of LL3 larvae (w1118 and Axn127) fed with the (Fig. 4 A–C). A simple model to explain this conundrum is that on indicated proteasome inhibitors, or DMSO as a negative control, under a Wg signaling activation, β-catenin is already stabilized, and the dissecting microscope. (B) Chromatographic separation of lipids from larvae 1118 127 effect of BTZ is mediated by modulation of other target(s) that (w and Axn ) at LL3 stage fed with BTZ (lanes 3 and 4) or DMSO can suppress Wg activity. In support of this model, a previous (control; lanes 1 and 2) using TLC analysis. Lane 5 shows the lipid standard, a study has shown that the proteasome inhibitor MG132 stabilizes mixture of monoglyceride (MG), diglyceride (DG), TG, and stearic acid (rep- β α α resenting FFA). Lipid levels were quantified using ImageJ; the relative FFA/ both -catenin and -Cat, and that the accumulation of -Cat TG ratios, shown below the image, were obtained by dividing the FFA/TG inhibits the transcriptional activity of β-catenin during chondrocyte ratios of different samples by the FFA/TG ratio of WT fed with DMSO. (C and differentiation (70). Thus, we evaluated the effect of BTZ on D) Confocal images of fat body from Axn127 larvae fed with BTZ (D) or DMSO protein levels of Arm and α-Cat in Drosophila by Western blot (C) stained with Nile Red and DAPI. analysis. As shown in Fig. 4D, BTZ stabilized both Arm and α-Cat in WT larvae; however, in Axn127 mutants, BTZ increased the level of α-Cat but had a weaker effect on the already elevated level OPZ are peptide epoxyketones that can irreversibly inhibit pro- of Arm protein (Fig. 4D). These observations suggest different teasome activity; and MRZ has a β-lactone backbone and is an 127 effects of BTZ on Arm and α-Cat proteins in Axn larvae, which irreversible proteasome inhibitor (59). As shown in Fig. 3A,IXZ may explain how BTZ suppresses Wg signaling. showed potent effects similar to BTZ by rescuing the fat defects in α 127 127 If increased -Cat by BTZ is responsible for the rescue of the Axn mutants. DLZ also suppressed the fat defects in Axn Axn127 defects in fat body, then we would expect to find that mutants (Fig. 3A), although its rescue effect was weaker than that overexpression of α-Cat in the fat body could rescue the Axn127 of BTZ and IXZ. In contrast, CFZ, MRZ, and OPZ did not rescue defects. To validate this model, we ectopically expressed α-Cat in μ the fat defects even at higher concentrations, such as 4 M(Fig. fat bodies by SREBP-Gal4 in the Axn127 background. We ob- 3A and Fig. S8). At the cellular level, CFZ, OPZ, and MRZ also served that fat body defects were strongly rescued in Axn127 127 failed to rescue the adipocyte defects in Axn mutants (Fig. S9). larvae with α-Cat overexpression (Fig. 4G) compared with the We also screened several chemical compounds that have been Axn127 larvae (Fig. 4F) and Axn127/+ larvae (Fig. 4E) as the reported to target the protein degradation process, including controls. At the cellular level, the sizes of the adipocytes were b-AP15, celastrol, degrasyn, epigallocatechin gallate, ONX-0914/ less heterogeneous in Axn127 larvae with α-Cat overexpression PR-957, PI-1840, VR23, and zinc protoporphyrin (60–67), and (Fig. 4G′, compared with Axn127 shown in Fig. 4F′) accompanied found that none of these compounds showed any obvious effects by increased accumulation of TG (Fig. 4H and Fig. S12). Taken on the fat defects in Axn127 mutants (Fig. S10). Taken together, together, these results suggest that BTZ may rescue the Axn127 our chemical screening results show that only peptide boronic defects through stabilization of α-Cat, and that overexpression of acids rescued the defective fat metabolism in Axn127 mutants. α-Cat is sufficient to rescue the Axn127 defects.

Zhang et al. PNAS | Published online August 21, 2017 | E7473 Downloaded by guest on September 23, 2021 Fig. 4. BTZ inhibits the expression of genes activated by the Wg signaling pathway and stabilizes α-Cat. (A–B′) The levels of nkd-lacZ expression are assayed by β-Galactosidase staining in guts from LL3 larvae of the indicated genotypes, fed with either DMSO (A and B) or BTZ (A′ and B′). (C) Quantification of transcript expression for arm, nkd, and Notum from larvae of the indicated genotypes and treatments. Comparisons of gene expression between BTZ-fed Axn127 mutants and DMSO-fed w1118 larvae are denoted by *; comparisons of gene expression between BTZ-fed Axn127mutants and DMSO-fed Axn127 larvae are denoted by +.(D) Western blot analyses of the effect of BTZ on the expression of Arm and α-Cat in Axn127 mutant and w1118 larvae. Actin is used as a loading control. (E–G) Images of LL3 larvae of the indicated genotypes: (E) SREBP-Gal4/+;Axn127/+,(F) SREBP-Gal4/+;Axn127, and (G) SREBP-Gal4/UAS- α-Cat- EGFP; Axn127. (Scale bar in E: 1.0 mm.) (E′–G′) Confocal images of fat body from larvae of the indicated genotypes (E–G) stained with Nile Red and DAPI. (Scale bar in E′:20μm.) (H) Quantification of TG levels from larvae of the indicated genotypes in E–G.*P < 0.05; +P < 0.05; **P < 0.01; ++P < 0.01; ***P < 0.001.

Inhibition of Wg Activity by BTZ Is Dependent on α-Cat. The data results show that the suppressive effects of BTZ and IXZ on presented above suggest the following model: BTZ may stabilize Axn127 mutants are dependent on α-Cat. α-Cat, which sequesters or inhibits the extra Arm caused by the Axn mutation, thereby attenuating the hyperactivation of Wg Effects of α-Cat and BTZ Treatment on Arm Distribution. Because el- 127 signaling and rescuing the defective TG accumulation (Fig. 5A). evated Wg signaling caused the adipocyte defects in Axn mu- To test whether α-Cat plays a critical role in mediating the strong tants (Figs. 2 and 4), we investigated whether the distribution of α suppressive effects of peptide boronic acids on the defects of the Arm in adipose tissue is affected by -Cat overexpression or BTZ Axn127 mutants, we generated Drosophila strains with α-Cat de- treatment. Using the larvae of the same genotypes described in 127 Fig. 4 E–G, we first analyzed the distribution of Arm in adipocytes pleted in the fat bodies of the Axn mutant larvae and then fed when α-Cat is increased. As shown in Fig. 6A, Arm has a punctate them BTZ, IXZ, or DMSO. Axn127 mutant larvae fed with either distribution under the plasma membrane of adipocytes in the BTZ or IXZ rescued the fat defects (Fig. 5 D and I) compared 127 controls. In contrast, the distribution of Arm is heterogeneous in with the Axn fed with DMSO as a control (Fig. 5C). Although 127 α Axn mutants; punctate Arm distribution is observed only along depletion of -Cat in the fat bodies of the WT larvae had little the plasma membrane of larger adipocytes, whereas Arm is uni- effect on their fat accumulation (Fig. 5 E, G, and J), depletion of formly distributed within the cytoplasm of the smaller adipocytes α 127 -Cat in the Axn mutant background (Fig. 5F) led to slightly (Fig. 6B). However, this heterogeneous distribution of Arm in 127 more severe fat defects than seen in Axn mutants (compared Axn127 mutant adipose tissue is almost normalized when α-Cat is with Fig. 5C, quantified in Fig. 5L). More importantly, neither expressed ectopically (compare Fig. 6C with the control shown in BTZ nor IXZ was able to rescue the fat defects of α-Cat–depleted Fig. 6B). These observations suggest that α-Cat overexpression is 127 127 Axn mutants (Fig. 5 H and K)comparedwithAxn as controls sufficient to restore the distribution of Arm. (Fig. 5 D and I). We also found that the phenotypes of fat body We next tested whether the α-Cat–dependent rescue effects of from whole larvae correlated with their TG levels (Fig. 5L). These BTZ on Axn127 mutants (Fig. 5) is correlated with the distribution

E7474 | www.pnas.org/cgi/doi/10.1073/pnas.1621048114 Zhang et al. Downloaded by guest on September 23, 2021 level of Arm was significantly elevated in fat bodies from Axn127 PNAS PLUS mutant larvae (with DMSO), especially in small adipocytes (Fig. 6E). The Arm level in w1118 larvae with BTZ treatment was mildly increased, but the distribution of Arm was not obvi- ously affected (Fig. 6F). Consistent with its rescue effects, BTZ treatment of Axn127 larvae strongly restored the punctate distri- bution of Arm at the adipocyte cortex (compare Fig. 6G with Fig. 6E). Compared with the WT larvae (Fig. 6D), depletion of α-Cat in the fat bodies did not alter the distribution of Arm in the control larvaefedwithDMSOorBTZ(Fig.6H and I). Arm staining also was markedly enhanced in fat bodies from the α-Cat–depleted Axn127 larvae fed with DMSO (Fig. 6J). Consistent with the results from whole larvae and TG levels (Fig. 5 H and L), BTZ treatment could no longer restore the Arm levels and distribution in the fat bodies when α-Cat was depleted in Axn127mutants (Fig. 6K). The sizes of adipocytes in these BTZ-treated larvae were also hetero- geneous (Fig. 6K). Taken together, these observations suggest that at the cellular level, α-Cat mediates the effects of BTZ by nor- malizing the distribution of Arm, which allows the rescue of Axn127 mutant phenotypes. CELL BIOLOGY

Fig. 5. Inhibition of Wg activities by BTZ is dependent on α-Cat. (A)Model: peptide boronic acids stabilize α-Cat, which sequesters or inhibits the extra Arm caused by the Axn mutation, thereby attenuating Arm-stimulated gene expres- sion and rescuing the defective TG accumulation caused by hyperactivation of the Wg signaling. α-Cat, α-catenin; APC, adenomatous polyposis coli protein; Arm, Armadillo;Arr,Arrow;Axn,Axin;Dsh,Dishevelled;Fz,;Lgs,Legless;Pan, Pangolin (homolog of TCF); Pygo, Pygopus; Sgg, Shaggy (GSK3 homolog); Wg, Wingless (Wnt homolog). Arrows indicate activation; blunt arrows, inhibition. (B–K) Representative images of LL3 larvae of the indicated genotypes and treatments: (B) w1118;(C, D,andI) Axn127.(E, G,andJ) FB-Gal4/+;UAS-α-CatRNAi/+. (F, H,andK) FB-Gal4; Axn127/UAS-α-CatRNAi,Axn127.(L) Quantification of TG from larvae of the indicated genotypes and treatments shown in B–K (n = 3 for each genotype). Comparisons between inhibitor-fed mutants and DMSO-fed WT lar- vae are denoted by *, comparisons between inhibitor-fed Axn127 mutants and Fig. 6. Effects of α-Cat and BTZ treatment on the distribution of Arm. (A–C) DMSO-fed Axn127 mutant larvae are denoted by +, comparisons between Confocal images of fat bodies from larvae of the indicated genotypes inhibitor-fed mutants and DMSO-fed FB>α-CatRNAi; + larvae are denoted by #, stained with Arm antibody (green) and DAPI (blue). (Scale bar in C:20μm.) and comparisons between Axn127 and FB>α-CatRNAi;Axn127” larvae are denoted Genotypes: (A) SREBP-Gal4/+;Axn127/+;(B) SREBP-Gal4/+;Axn127;(C) SREBP- ++ by §. *P < 0.05; #P < 0.05; P < 0.01; ***P < 0.001; ###P < 0.001; §§§P < 0.001. Gal4/UAS-α-Cat-EGFP; Axn127. These images were taken under the same fixation, staining and microscopic settings. (D–K) Confocal images of adi- pocytes from larvae of the indicated genotypes and treatments stained with pattern of Arm in adipocytes using immunostaining. In the anti-Arm antibody (red) and DAPI (blue). These images were taken under the 1118 control (w with DMSO), low levels of Arm exhibited a same conditions. Genotypes: (D and F) w1118;(E and G) Axn127;(H and I) FB- punctate localization at the cortex of adipocytes (Fig. 6D). The Gal4/+;UAS-α-CatRNAi/+;(J and K) FB-Gal4; Axn127/UAS-α-CatRNAi,Axn127.

Zhang et al. PNAS | Published online August 21, 2017 | E7475 Downloaded by guest on September 23, 2021 Discussion There are two potential mechanisms behind the elevation of Hyperactivated Wnt signaling can promote a variety of human α-Cat levels by BTZ to rescue the hyperactivated Wg signaling in 127 cancers, in addition to diseases related to dysregulated cellular Axn mutants. First, α-Cat may sequester the extra Arm/β-cat- 127 metabolism, such as diabetes (1–4, 11, 14, 71). The identification enin in the cytoplasm in Axn mutants, thereby preventing cyto- of drugs that target Wnt signaling for therapeutics remains a plasmic Arm from translocating to the nucleus and activating gene α challenge, owing in part to the lack of an efficient in vivo expression. Second, -Cat may enter into the nucleus and directly screening system. In this study, we established a Drosophila inhibit Arm-dependent gene expression. These two mechanisms model to study the effects of hyperactivated Wnt signaling and are not mutually exclusive, and our present data cannot distinguish then explored its use in screening for small molecules targeting between these two possibilities. α Wnt signaling in vivo. Specifically, we characterized adipocyte Interestingly, our results indicate that BTZ stabilizes -Cat, but β 127 127 defects in Axn127 mutants, and our genetic analyses suggest that not -catenin, in Axn mutants, unlike in Axn larvae fed with these phenotypes are regulated by the canonical Wnt signaling DMSO. This phenomenon could be due to different degradation α β β pathway. Making use of the advantages of Drosophila as a plat- mechanisms of -and -catenins. Degradation of -catenin de- pends on the β-catenin destruction complex (8). Because the Arm/ form for in vivo chemical screens, we found that the adipocyte 127 127 β-catenin destruction complex is compromised in Axn mutants, defects were rescued when Axn mutants were fed with peptide β boronic acids, such as BTZ, DLZ, and IXZ. Further analyses ubiquitination of Arm/ -catenin and subsequent proteasome- suggest that BTZ decreases Wnt signaling activities, and that its dependent degradation are already less efficient, thereby re- rescue effects are dependent on α-Cat. These results establish a ducing the response to additional inhibition by BTZ. This expla- nation is consistent with our findings showing that Arm is strongly proof of concept for using this model system to identify and 127 optimize drugs for treating diseases caused by hyperactivated increased in Axn mutants but is not further increased by BTZ. α Axn127 Wnt signaling. In contrast, -Cat expression is further increased in mu- tants by BTZ. This difference may account for the effects of BTZ α 127 Chemical Inhibition of Wnt Signaling Activities by Stabilizing α-Cat. in stabilizing -Cat and inhibiting Wnt signaling in Axn mutants. Drosophila models mimicking such human diseases as cancer, BTZ is an FDA-approved (in 2003) drug for treating multiple diabetes, and neurodegeneration have been used extensively for myeloma and mantle cell lymphoma (59, 78). The exact mecha- chemical screens and drug discovery (28–31). Axn127 mutants nism of how BTZ kills cancer cells remain unclear, however. may serve as an experimental system to screen small molecules Despite the impressive efficacy of BTZ in treating hematologic that can suppress hyperactivated Wnt signaling or rescue visible malignancies and certain types of lymphoma, the outcomes of defects in fat bodies. Drosophila offers the general advantages of BTZ treatment for solid tumors have been variable, for unknown a screening model with cost efficiency, facilitated use, and ca- reasons (79). In a phase I clinical trial of 102 patients with relapsed pability for direct in vivo tests. Moreover, the phenotypes of or refractory colorectal cancer, BTZ treatment was not effective in 127 patients with advanced colorectal cancer (80). One possible ex- Axn larvae are robust, easy to score, and sensitive to changes planation for this finding is that BTZ is less permeable in co- in Wg activity. Our identification and analysis of BTZ and two lorectal cancers than in other types of tumors. Our results suggest other peptide boronic proteasome inhibitors as Wnt signaling that BTZ inhibits Wnt signaling activity by stabilizing α-Cat, and inhibitors in vivo provide a proof of principle for the application that this inhibitory effect is more effective when the degradation of of this system to identify and evaluate additional small molecules β-catenin is compromised. If the mechanism that we observed in targeting hyperactivated Wnt signaling. Interestingly, three other Drosophila is conserved in mammalian cells, it will be interesting proteasome inhibitors (CFZ, MRZ, and OPZ) did not show any to further screen cancer cells with hyperactive Wnt activity and to Axn127 obvious rescuing effects in mutants at different concen- test whether BTZ can stabilize α-Cat in these cancer cells. Fur- trations. CFZ, MRZ, and OPZ are irreversible proteasome in- thermore, given that hyperactive Wnt activity from overexpression hibitors with different structures compared with the peptide of Wnt10 or β-catenin causes severe defects in adipocyte boronic acids (59). Further studies are warranted to explore the tissues, it will be interesting to investigate whether mice with underlying molecular mechanisms of these proteasome inhibitors. β β compromised -catenin destruction complex also show adipocyte Because -catenin is degraded by the ubiquitination-proteasome defects, and if so, whether this phenomenon will respond to BTZ. pathway, it seems puzzling that proteasome inhibitors can down- 127 It is surprising that none of the compounds that have been regulate Wg signaling activities in Axn mutants. Several studies reported to inhibit Wnt signaling worked in our system, espe- have shown that proteasome inhibitors MG132 and BTZ stabilize β β cially the iCRTs, which are known to work in both Drosophila -catenin and up-regulate -catenin/TCF-dependent gene expres- S2 cells and mammalian cells (58). We could not detect any ef- sion (70, 72, 73); however, it also has been reported that protea- fects when feeding larvae with these compounds. There are many β some inhibition decreases -catenin/TCF transcriptional activities possible explanations for these findings, considering the differ- in hepatocellular carcinoma cells (74). These differences may be ences between our system and the approaches used by Gonsalves related to the different biological contexts and cell lines used in et al. (58). Perhaps the iCRTs and other known Wnt signaling these studies. Alternatively, proteasome inhibitors may affect the inhibitors do not offer a sufficient therapeutic window to reduce stability of multiple components of the Wnt signaling pathway Wg signaling sufficiently to rescue the Axn127 fat body defects rather than exclusively targeting β-catenin. Our genetic and bio- 127 without preventing larvae growth or development as a conse- chemical analyses of Axn mutants suggest that BTZ suppresses quence of Wg signaling inhibition in vivo. Wg signaling activities by stabilizing α-Cat. This model is consis- tent with previous reports indicating that the overexpression or Functions of Wnt Signaling in Regulating Adipocyte Biology in Drosophila. stabilization of α-Cat can suppress Wnt signaling activities by an- Wnt signaling inhibits adipogenesis in a cultured mouse pre- tagonizing β-catenin/TCF-dependent transcription in Xenopus adipocyte 3T3-L1 cell experimental system (20). Overexpression of embryos, chondrocytes, and L cells, whereas the loss of α-Cat in- β-catenin in mouse adipocyte progenitors led to compromised adi- creases TCF-dependent gene expression in colon cancer cell lines pocyte differentiation and development, but no obvious defects (70, 75–77). Our results show that stabilization of α-Cat by peptide were found when β-catenin was overexpressed in mature adi- boronic inhibitors is sufficient to down-regulate Wg activities in pocytes (19). Thus, these and many other studies have estab- Axn127 mutants, suggesting that the differential effects of BTZ on lished that Wnt signaling inhibits adipogenesis through a Arm and α-Cat levels may be the key to determining the inhibitory canonical β-catenin/TCF–dependent signaling transduction in effects of BTZ on Wg signaling. mammals (14, 81, 82). Compared with the role of Wnt signaling in

E7476 | www.pnas.org/cgi/doi/10.1073/pnas.1621048114 Zhang et al. Downloaded by guest on September 23, 2021 regulating adipogenesis, less is known about the role of Wnt sig- Crosstalk Between Different Tissues with Hyperactivated Wnt Signaling. PNAS PLUS naling in regulating fat accumulation in adipocytes, which is de- Multiple tissues and organs are involved in the maintenance of fat termined by the net effect of fat synthesis (lipogenesis and TG homeostasis, including adipose tissue, gut, liver, and muscle. Pre- synthesis) and fat breakdown (lipolysis and subsequent fatty acid vious studies in larvae and adult flies have identified the intestine oxidation). This is likely related to the fact that these processes as a key organ in lipid uptake and metabolism (42, 89, 90). Con- occur during adipogenesis, making it challenging to clearly dissect sistent with those studies, expression of WT Axn in either the gut the roles of Wnt signaling in regulating both processes in mam- or the fat body alone partially rescued the adipocyte defects in malian cells. Axn127 mutant larvae, but the adipocyte defects were more strongly Drosophila provides a unique system for studying fat metab- rescuedwhenWTAxnwasrestoredinboththefatbodyandthe olism in which the different processes pertinent to adipocyte gut. This finding suggests that both the fat body and the gut are biology occur at different developmental stages. For example, important factors in the fat body defects in Axn127 larvae. Despite lineage studies show that adipogenesis in Drosophila occurs only these observations, our clonal analyses of Axn mutant alleles in fat during embryogenesis (83). Lipogenesis occurs only during larval bodies suggest that a gain of Wg activity within adipocytes can development in Drosophila fat body, which is functionally anal- affect lipid metabolism, indicating a cell-autonomous effect of Wg ogous to the mammalian adipose tissue and liver (84, 85). During signaling in fat metabolism. Further studies are needed to more the larval stages, adipocytes in the fat body increase in size in clearly define the specific functions of Wg signaling in regulating concert with fat accumulation, but not in number, because the lipid metabolism at the cellular and organismal levels. nuclei of adipocytes undergo endoreplication cycles without In conclusion, our study illustrates that hyperactive Wg sig- nuclear division (43, 86). Lipolysis and fatty acid oxidation occur naling disrupts fat metabolism in Drosophila larvae, and that this mainly in pupae, providing energy and metabolic intermediates defect can be rescued by peptide boronic acids in an α-Cat–de- 127 required for metamorphosis during the pupal stage. pendent mechanism. Our results show that Axn mutant larvae The morphology of fat bodies in Axn127 mutant larvae appears represent an attractive system for screening and optimizing small normal before the early third instar, and defects start to become molecules that target Wnt signaling in vivo. This study suggests apparent only at mid-third instar, accompanied by a significant that pharmacologic stabilization of α-Cat may provide an at- increase in FFAs and reduction in TGs. These observations favor tractive strategy for attenuating Wnt signaling. a scenario in which the processes involved in fat accumulation, including fatty acid and TG synthesis, lipolysis, and fatty acid Materials and Methods oxidation instead of adipogenesis, are defective in the Axn127 Detailed descriptions of the materials and methods used in this study, in- CELL BIOLOGY mutants. Given that endoreplication is correlated with increased cluding Drosophila stocks (32, 36); chemical inhibitors; Western blotting and cell size and fat accumulation in fat body adipocytes, it will be immunostaining (91); quantitative RT-PCR (92); fat body staining with BODIPY, Nile Red, DAPI, and phalloidin; quantification of TG levels by Oil interesting to further characterize the relationships among Wnt Red O staining and a TG quantification colorimetric kit (92); mass spec- signaling, endoreplication, adipogenesis, and fat metabolism. trometry analysis of lipids; TLC (93); whole-animal drug screens; and statis- 127 Another interesting phenotype of Axn mutant larvae is that tical analyses, are provided in SI Appendix. the defects in the fat bodies appear to be heterogeneous at the organismal and cell levels. The defects are stronger in abdominal ACKNOWLEDGMENTS. We thank Hui Jiang and Daniel Ory at the Washing- fat bodies compared with fat bodies in the anterior and posterior ton University Metabolomics Facility for the mass spectrometry analysis of parts. At the cellular level, most of the adipocytes within mutant lipids; Yashi Ahmed and the Bloomington Drosophila Stock Center (National Institutes of Health Grant P40OD018537) for Drosophila strains; and the abdominal fat bodies fail to accumulate fat and remain small and Developmental Studies Hybridoma Bank at the University of Iowa for mono- intermixed with a few large adipocytes during late third instar. clonal antibodies. We also thank Sarah Bondos and Margrit Schubiger for Unlike mammals, in which the heterogeneity of adipocytes has critical comments on the manuscript, and Craig Kaplan, Keith Maggert, and been well appreciated (87), the physiological significance of ad- Fajun Yang for stimulating discussions. This work was supported by grants from the National Institutes of Health (DK095013, to J.-Y.J., and ipocyte heterogeneity in Drosophila remains poorly understood, GM120046 and CA149275, to W.D.) and the Chicago Biomedical Consortium despite the original observations reported decades ago (88). (to W.D.).

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