Diabetes Volume 67, December 2018 2569

Pygo2 Regulates Adiposity and Glucose Homeostasis via b-Catenin–Axin2–GSK3b Signaling Pathway

Yuan-Yuan Xie,1,2 Chun-Li Mo,1,2 Yi-Huang Cai,1,2 Wen-Jie Wang,1,2 Xin-Xin Hong,1,2 Kun-Kun Zhang,1,2 Qing-Feng Liu,1,2 Yun-Jia Liu,1,2 Jing-Jing Hong,1,2 Ting He,1,2 Zhong-Zheng Zheng,1,2 Wei Mo,1,2 and Bo-An Li1,2

Diabetes 2018;67:2569–2584 | https://doi.org/10.2337/db18-0311

Wnt/b-catenin signaling plays a key role in regulating types of cancers and has become a major public health adipogenesis through indirectly inhibiting the expression problem in industrialized countries (1,3). It is accepted of C/EBPa and peroxisome proliferator–activated recep- that obesity results from a positive imbalance between tor g (PPARg); however, the detailed molecular mecha- energy intake and output. Two potential mechanisms ex- nism remains poorly understood. Moreover, the factor(s) plain the development of metabolic complications resulting INLTRANSDUCTION SIGNAL that determines the Wnt/b-catenin output level during from obesity, including 1) lipotoxicity, where excess lipids fi adipogenesis is also not completely de ned. In this overflow from an incompetent and dysfunctional adipose study, we showed that Pygo2 exhibited a declined expres- tissue (4), and 2)specific patterns of adipokines and proin- sion pattern during adipocyte differentiation, result- flammatory cytokines released from dysfunctional adipose inginanattenuatedWnt/b-catenin output level. The tissue (2). These raise a new question: why do adipocytes mechanism study indicated that Pygo2 inhibition led to change from a healthy storage tissue to one that seemingly the downregulation of Axin2, a constitutive Wnt target, in promotes the metabolic syndrome? Indeed, the “adipose the cytoplasm. Consequently, Axin2-bound GSK3b was expandability hypothesis” (5,6) suggests that the total released and translocated into the nucleus to phosphor- fi ylate C/EBPb and Snail, resulting in an increase in the adipose tissue in an individual may have a maximal xed DNA binding activity of C/EBPb and decreased protein capacity for safely storing fat. This capacity may be de- stability of Snail, which subsequently activated the ex- termined by multiple factors, including the number of pre- pression of C/EBPa and PPARg. Consistent with this, existing preadipocytes, genetic programs of adipogenesis, embryonic fibroblasts from Pygo22/2 mice exhibited spon- programs of vasculogenesis, or functionality of other cellular taneous adipocyte differentiation, and adipocyte precursor– components within the adipose tissue. Therefore, under- specific Pygo2-deficient mice exhibited increased adiposity standing the molecular mechanisms that regulate the above with decreased energy expenditure. We further showed factors might provide novel strategies to prevent obesity impaired glucose tolerance and decreased systemic insulin and related metabolic complications. The study of adipo- sensitivity in Pygo2-deficient mice. Our study revealed an genesis has been greatly facilitated by the development of association between Pygo2 function and obesity or diabetes. immortalized preadipocyte lines, such as 3T3-L1 and 3T3- F442A cells (7–10), that differentiate into adipocytes and recapitulate many metabolic and endocrine functions of Adipose tissue plays crucial roles in the regulation of energy adipocytes in vivo. In response to stimulators of adipo- metabolism through storing and releasing fuel as an energy genesis, two transcription factors, C/EBPb and C/EBPd,are depot and through secreting hormones and cytokines as an transiently and rapidly induced. These proteins stimulate endocrine organ (1,2). Obesity can increase the risk of expression of key adipogenic transcription factors, namely, diabetes, hypertension, cardiovascular diseases, and several C/EBPa and peroxisome proliferator–activated receptor g

1State Key Laboratory of Cellular Stress Biology, Innovation Center for Cell This article contains Supplementary Data online at http://diabetes Signaling Network, School of Life Sciences, Xiamen University, Xiamen, Fujian, .diabetesjournals.org/lookup/suppl/doi:10.2337/db18-0311/-/DC1. China Y.-Y.X. and C.-L.M. contributed equally to this work. 2Engineering Research Center of Molecular Diagnostics, Ministry of Education, © 2018 by the American Diabetes Association. Readers may use this article as School of Life Sciences, Xiamen University, Xiamen, Fujian, China long as the work is properly cited, the use is educational and not for profit, and the Corresponding author: Bo-An Li, [email protected]. work is not altered. More information is available at http://www.diabetesjournals Received 14 March 2018 and accepted 13 September 2018. .org/content/license. 2570 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018

(PPARg), which then work together to induce the expression PDGFRa-Cre mice were purchased from The Jackson Lab- of the adipocyte-specific genes necessary to produce the oratory (stock no. 013148) and then were back-crossed adipocyte phenotype (11,12). Identification of new factors into the FVB background for more than six genera- that regulate progression through this adipogenic program is tions. For adipocyte precursor–specific deletion of Pygo2, fl fl crucial for us to gain new insight into this biological process. PDGFRa-Cre mice were crossed with Pygo2 ox/ ox mice fl Wnt/b-catenin signaling is crucial in cell proliferation to generate PDGFRa-Cre;Pygo2 ox/+ mice, which were fl fl and differentiation throughout vertebrate embryonic de- then intercrossed to generate PDGFRa-Cre;Pygo2 ox/ ox 2 2 velopment and tumorigenesis (13–15). b-Catenin plays (Pygo2pre / ) mice. Genetically obese (ob/ob)micewere a central role as a transcriptional coactivator in this process. purchased from Shanghai SLAC Laboratory Animal Co., In the absence of upstream signaling stimulation, cytoplas- Ltd., and the origin is 000632-B6.Cg-Lepob of The Jackson mic b-catenin is phosphorylated by glycogen synthase ki- Laboratory. Animals were maintained on a 12-h light/12-h nase 3b (GSK3b) in a complex containing Axin and the dark cycle in a temperature-controlled barrier facility with tumor suppressor adenomatous polyposis coli and is tar- free access to water and food. Age-matched male mice were geted for ubiquitin-mediated proteasomal degradation. used for all experiments for uniformity and to avoid the Stimulation by upstream signaling leads to the inhibition hormone impact of females. Animal studies were approved of phosphorylation and degradation of b-catenin, which by the Institutional Animal Care and Use Committee of subsequently translocates into the nucleus and binds to the Xiamen University. lymphoid-enhancing factor 1/T-cell factor (Lef1/Tcf) family of transcription factors. In turn, the b-catenin–Lef1/Tcf Blood Analysis complex regulates the expression of downstream target For glucose tolerance test (GTT), we deprived the mice of genes involved in diverse cellular processes (16,17). Previous food for 14 h and then injected them intraperitoneally studies have shown that Wnt/b-catenin signaling could with 2 g glucose per kilogram body weight. For insulin suppress adipocyte differentiation through inhibition of tolerance test (ITT), mice were deprived of food for 4 h C/EBPa and PPARg (18,19). Although a subset of Wnt/ with ad libitum access to water and then they were injected b-catenin signaling components have shown to be regulated intraperitoneally with 0.75 units human insulin per kilo- during induction of adipogenesis (19–22), additional Wnt/ gram body weight. We collected blood samples and mea- b-catenin components that govern Wnt/b-catenin activ- sured the glucose concentration with a glucometer (One ity in this process have yet to be defined. Moreover, as TouchUltra;LifeScanInc.,Milpitas,CA).Thelevelof b-catenin usually acts as a transcriptional activator, the serum insulin was determined using a mouse ELISA kit mechanism by which b-catenin signaling blocks C/EBPa (ALPCO,Salem,NH). and PPARg gene expression is still not clear. MRI Experiments fi Drosophila genetic studies have identi ed Pygopus The body compositon of mice was performed on a small (Pygo) as a nuclear component of Wnt/b-catenin signaling animal composition analyzer (MesoMR21-60H-I; Shang- (23,24). Pygo proteins (Pygo1 and Pygo2 in mammals) are hai Niumag Corporation, Shanghai, China) with the fol- thought to promote b-catenin-Lef1/Tcf transcriptional lowing parameters: SFO1 (MHz) = 21.77, P1 (us) = 11, P2 activation through the regulation of b-catenin nuclear (us) = 22, and SW (KHz) = 20. MR images were acquired on retention and/or by facilitating b-catenin to recruit coac- a 0.55 T MRI Instrument (MesoMR23-060H-I; Shanghai – tivators (25 31). Recent studies have revealed that Pygo2 Niumag Corporation). The T1 weighted imaging parame- mediates b-catenin activity in a gene- and tissue-depen- ters were customized as follows: SFO1 (MHz) = 23.31, FOV dent manner in mammalian cells (31,32). We have pre- Read (mm) = 100, FOV Phase (mm) = 100, Matrix = 256 3 viously reported the function of Pygo2 in mammary gland 256, TR (ms) = 400, TE (ms) = 18, eight slices, and a slice development (31), breast cancer stem cells (30), and breast thickness of 2 mm with in-plane resolution of 0.4 3 cancer chemoresistance (33). In the current study, we 0.4 mm (voxel size). Mice were anesthetized with isoflur- fi showed that Pygo2 is a newly identi ed activator of ane to ensure stability throughout the experiment. Wnt/b-catenin signaling in the regulation of adipocyte differentiation. Furthermore, we identified a mechanism Fraction of Adipose Tissues that mediates the inhibition of C/EBPa and PPARg ex- The white adipose tissue (WAT) was from inguinal adipose pression by Pygo2 and b-catenin during adipogenesis. Our depots of 12-week-old mice and human subjects that study reveals an association between C/EBPa/PPARg ex- underwent elective thigh surgery. We washed and minced pression and Wnt/b-catenin activation, which highlights WAT with PBS, digested for 1 h at 37°C with Type II the role of Wnt/b-catenin in the regulation of adiposity collagenase (C6685-1G; Sigma-Aldrich) solution, passed and glucose homeostasis. the mixture through a nylon filter (70 mm) to remove undigested materials, and centrifuged at 1,200 rpm for RESEARCH DESIGN AND METHODS 10 min at 4°C. We recovered the floating mature adipo- Animals cytes and the pelleted stromal vascular fraction (SVF) The generation and genotyping of null and floxed Pygo2 respectively. Human adipose tissue was collected after alleles were performed as previously described (34). informed consent and in agreement with the Institutional diabetes.diabetesjournals.org Xie and Associates 2571

Review Board of The First Affiliated Hospital of Xiamen Chromatin Immunoprecipitation University. Chromatin immunoprecipitation (ChIP) was performed using a ChIP Assay Kit according to its biotechnology Lentivirus-Mediated Gene Transfer, RNA Interference, or Gene Knockout by CRISPR/Cas9 protocol (Upstate). In brief, chromatin samples were pre- b The lentiviral pBobi vector was used to express hemag- pared and immunoprecipitated with IgG, C/EBP anti- b body (sc-150; Santa Cruz Biotechnology), and protein A/G glutinin (HA)-Pygo2, -catenin, dnTCF4 (dominant- fi negative TCF4, a b-catenin binding domain deletion beads. Puri ed DNA was subjected to real-time qPCR fl a g mutant), and Axin1. Axin2 and Axin2-DGSK3b (Axin2 with primers anking the C/EBP and PPAR promoter b mutant without GSK3b binding domain 353–410) were sequences of the C/EBP binding sites. Supplementary cloned into the lentiviral vector pLV-CS2.0. The lentiviral Table 4 lists the primer sequences. pLL3.7 vector was used to express short hairpin RNA Histological Studies (shRNA) directed against mouse Pygo2, Axin2,oranon- Pieces of epididymal WAT from the mice were fixed in 10% silencing scrambled control sequence. The target sequen- neutral buffered formalin, dehydrated in ethanol, embed- ces were provided in Supplementary Table 1. b-Catenin ded in paraffin, and cut at a thickness of 4 mm. Sections knockout or Pygo2 knockout 3T3-L1 cells were con- were deparaffinized, rehydrated, and stained with hema- structed with lentiCRISPRv2 vector by CRISPR/Cas9 toxylin-eosin. The adipocyte size was quantified using technique. The target site was designed as follows: Image-Pro Plus software, and at least 600 cells per sample b-catenin 59-CAGCGTCAAACTGCGTGGAT-39 and were measured. Pygo2 59-CCTGCGCCCCCCACTTTAG-39. The generation of the lentivirus was performed by cotransfecting pBobi, Statistical Analysis pLV-CS2.0, pLL3.7, or lentiCRISPRv2 carrying the expres- Each experiment was repeated at least three times. Rep- sion cassette with helper plasmids pVSV-G and pHR into resentative experiments are shown unless stated other- wise. Data were analyzed using the GraphPad software HEK 293T cells using Turbofect reagent (Thermo Fisher 6 Scientific, Waltham, MA). The viral supernatant was col- package. Data are presented as the means SD (in vitro lected 48 h after transfection. 3T3-L1 cells were infected experiments) or SEM (in vivo experiments) and were analyzed by unpaired two-tailed Student t test. P , with viral supernatants containing 10 mg/mL Polybrene at fi 40–50% confluency and were given fresh medium 24 h later. 0.05 was considered statistically signi cant. All data points were included in the final analysis, without exclusions. Western Blotting and Antibodies No randomization was used and the experimenter was Western blotting was performed according to standard blinded to genotypes. procedures. The following antibodies were used: Pygo2, a rabbit polyclonal antibody used as previously described RESULTS (30); p-Ser96-Snail, a rabbit polyclonal antibody raised Expression of Pygo2 in Differentiating 3T3-L1 Cells and against a synthetic peptide (amino acids 89–105 with Adipose Tissue p-Ser96 modified); and p-Ser184-C/EBPb antibody (gift To investigate a potential role of Pygo proteins in adipo- from Xi Li, Biology Science Institutes, Chongqing Medical genesis, we first analyzed the Pygo1 and Pygo2 protein University, Chongqing, China) (35). The other commercially expression levels during differentiation of 3T3-L1 pre- available antibodies are listed in Supplementary Table 2. adipocytes into mature adipocytes. Interestingly, Western blotting demonstrated that the Pygo2 protein levels RNA Isolation and Real-time Quantitative PCR progressively declined in contrast to the levels of the Total RNA was isolated from cells or adipose tissues using adipocyte differentiation marker adipocyte fatty acid Tripure reagent (Roche, Basel, Switzerland). First-strand binding protein 4 (FABP4) after hormonal stimulation, cDNA was synthesized using a HiFi-MMLV cDNA Kit from whereas Pygo1 expression did not change significantly Beijing CoWin Biotech (CWBIO, Beijing, China). UltraSYBR (Fig. 1A). Consistently, semi-qPCR analysis revealed Mixture (CWBIO) was used for the real-time quantitative a similar expression pattern for Pygo1 and Pygo2 during PCR (qPCR) reaction, and the expression levels were differentiation (Fig. 1B). We next examined the distri- quantified using the DDCT (where CT is threshold cycle) bution of Pygo2 mRNA in mouse adipose tissue. As shown method. Primer sequences are provided in Supplementary in Fig. 1C, Pygo2 mRNA was expressed in the SVF of the Table 3. tissues containing committed preadipocytes and was Luciferase Assays essentially absent in mature adipocytes in subcutaneous 3T3-L1 cells in six-well plates were transfected with the adipose depots. A parallel experiment was also conducted indicated plasmids using Lipofectamine 3000 (Invitrogen, on a human sample, and a result similar to that in mouse Life Technology, Waltham, MA). The total amount of adipose tissue was achieved (Fig. 1D). We also examined plasmid DNA transfected was made equivalent by adding the expression levels of Pygo1 and Pygo2 in genetically empty vectors. Cells were harvested after 48 h and pro- obese ob/ob mice. The expression level of Pygo2, but not cessed for luciferase and b-galactosidase assays, and data Pygo1, was decreased in the WAT of ob/ob mice compared were normalized to b-galactosidase levels. with that in wild-type (WT) mice (Fig. 1E). These data 2572 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018

Figure 1—Pygo2 expression pattern in cultured 3T3-L1 cells and adipose tissue. A and B: 3T3-L1 preadipocytes were induced to differentiate with MDI for 7 days. Whole-cell lysates were extracted at the indicated days of differentiation. Protein levels of Pygo2, Pygo1, and the adipocyte differentiation marker FABP4 were detected by Western blotting. ERK1/2 was used as a loading control (A). Pygo2, Pygo1, and Fabp4 mRNA levels were determined by semi-qPCR during differentiation of 3T3-L1 (B). C: Pygo2 mRNA expression in the SVF and mature adipocytes (Ad) in subcutaneous adipose depots of 12-week-old mice (n = 3). The results were obtained by real-time qPCR (left) and semi-qPCR (right). D: Pygo2 mRNA levels were measured using real-time qPCR in subcutaneous SVF and mature adipocytes from five unrelated human subjects. E: Real-time qPCR analysis of the Pygo1 and Pygo2 mRNA levels in the epididymal WAT of WT and ob/ob mice at 8 weeks of age (n = 3, left), and Western blotting determined the Pygo2 expression (right). For C and E, each bar represents the average value obtained from three animals. For D, each bar represents the average value of three technical replicates. C–E: Error bars represent SEM. **P , 0.01; ***P , 0.001.

suggest that Pygo2 may play a role during adipocyte the full differentiation cocktail (MDI) for 7 days. As shown differentiation. in Fig. 2A, the efficient expression of Pygo2 during cell fi Ectopic Expression of Pygo2 Attenuates Adipocyte differentiation was con rmed by immunoblotting using Differentiation, Whereas Knockdown of Pygo2 anti-HA and Pygo2 antibodies. As expected, 3T3-L1 pre- Enhances the Differentiation Process and Induces adipocytes infected with a control lentivirus underwent Spontaneous Differentiation efficient differentiation, and fat accumulation was visual- The expression pattern of Pygo2 in preadipocytes and ized by staining lipids with Oil Red O. In contrast, Pygo2 adipose tissue suggested that overexpression of Pygo2 overexpression effectively inhibited the accumulation of might impair the differentiation process. To test this, lipid droplets (Fig. 2B), concomitant with reduced expres- 3T3-L1 cells were infected with a lentivirus carrying mouse sion levels of C/EBPa, PPARg, and FABP4 (Fig. 2C). These Pygo2 or a control lentivirus. These cells were then induced data indicate that Pygo2 may negatively regulate adipo- to differentiate into mature adipocytes in the presence of cyte differentiation. Therefore, knockdown of Pygo2 in diabetes.diabetesjournals.org Xie and Associates 2573

Figure 2—Forced expression of Pygo2 inhibits adipocyte differentiation, whereas knockdown of Pygo2 enhances the differentiation process and induces spontaneous differentiation. A–C: 3T3-L1 cells were infected with lentivirus expressing GFP or HA-Pygo2. Western blotting indicated protein expression of HA and Pygo2 (A). Then, the cells were induced to differentiate with MDI for 7 days and were stained with Oil Red O to visualize the degree of lipid accumulation (scale bar, 50 mm) (B). Western blotting indicated the expression level of C/EBPa, PPARg, and FABP4 (C). D and E: 3T3-L1 cells were stably expressed with control (Ctrl) shRNA or two different Pygo2 shRNAs and then induced with MDI for 4 days. Cells were stained with Oil Red O to visualize lipid drops. Scale bar, 50 mm(D). The expression levels of Pygo2 (day 0), as well as C/EBPa, PPARg, and FABP4 (day 4) were assessed by Western blotting (E). F: 3T3-L1 cells were stably expressed with Ctrl shRNA or two different Pygo2 shRNAs and maintained without MDI treatment. Two weeks after confluence, these cells were stained with Oil Red O. Scale bar, 50 mm. preadipocytes may enhance the differentiation process. To These observations further support our hypothesis that test this hypothesis, we infected 3T3-L1 preadipocytes Pygo2 negatively regulates adipocyte differentiation. with lentivirus expressing Pygo2 shRNAs or a control virus, and the infected cells were induced by MDI for Pygo2 Facilitates b-Catenin Signaling Function 4 days. As shown in Fig. 2D and E,knockdownof in 3T3-L1 Cells Pygo2 significantly accelerated the differentiation process Previous studies have indicated that Pygo2 promotes when examined by both Oil Red O staining and the b-catenin activity in a tissue- and gene-dependent manner expression of differentiation markers. When the infected in mammals. To directly assess whether Pygo2 facilitates cells were allowed to proliferate to confluence and main- b-catenin signaling in 3T3-L1 preadipocytes, we at first tained without MDI treatment for 2 weeks, knockdown of performed Lef1/Tcf luciferase reporter assays. Sole expres- Pygo2 was found to undergo spontaneous adipogenesis, sion of either b-catenin or Pygo2 in 3T3-L1 cells stimu- whereas the control cells failed to differentiate (Fig. 2F). lated the activity of SuperTOPFlash containing seven 2574 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018 optimal Lef1/Tcf binding sites. Coexpression of b-catenin distribution of GSK3b via the b-catenin–Axin2 cascade and Pygo2 resulted in a further increase in SuperTOPFlash during adipogenesis. To this end, we overexpressed activity (Fig. 3A). In contrast, knockdown of Pygo2 in 3T3- b-catenin or dnTCF4 in 3T3-L1 cells induced by MDI L1 cells resulted in significantly lower SuperTOPFlash for 36 h. As shown in Fig. 4B and C, overexpression of activity (Fig. 3B). Consistently, Axin2, a constitutive b-catenin resulted in increased cytoplasmic Axin2 and downstream target of Wnt/b-catenin signaling that steadily GSK3b but decreased nuclear GSK3b. In contrast, over- expresses in almost all Wnt/b-catenin–activated cells, expression of dnTCF4 resulted in decreased cytoplasmic showed increased or reduced expression in both mRNA Axin2 and GSK3b but increased nuclear GSK3b. When and protein levels upon Pygo2 overexpression or knock- we knocked down Pygo2 expression in 3T3-L1 cells down (Fig. 3C and D). Wnt/b-catenin signaling is rapidly and performed the same experiment, results similar to suppressed upon induction of 3T3-L1 cell differentiation those achieved with the overexpression of dnTCF4 in response to 1-methyl-3-isobutylxanthine (IBMX) were observed (Fig. 4D). Because GSK3b phosphorylates (19,36). We investigated whether knockdown of Pygo2 C/EBPb and Snail in nucleus, we investigated the im- substitutes for the IBMX function. As shown in Fig. 3E and pact of Wnt/b-catenin signaling and Pygo2 on C/EBPb F, when cells could not be effectively induced to differen- and Snail. Our results revealed that overexpression of tiate by omitting IBMX from the differentiation cocktail b-catenin in 3T3-L1 cells resulted in decreased phosphor- (DI), knockdown of Pygo2 led to higher lipid accumulation ylation on Ser184 of C/EBPb and Ser96 of Snail (Fig. 4E), and expression of the adipogenic markers than in control. whereas overexpression of dnTCF4 or knockdown of Moreover, we found that overexpression of dnTCF4 could Pygo2resultedinincreasedphosphorylationonthe not further enhance adipogenesis in the presence of Pygo2 same sites (Fig. 4F and G). Consequently, activation of shRNA under the DI condition (Fig. 3G and H). Our Wnt/b-catenin signaling by b-catenin overexpression results demonstrate that Pygo2 can inhibit differentiation resulted in decreased occupancy, whereas suppression of of 3T3-L1 cells through mediation of b-catenin signaling. Wnt/b-catenin signaling by dnTCF4 overexpression or Pygo2 knockdown resulted in increased occupancy of Pygo2 Regulates the Cytoplasmic-Nuclear Distribution the C/EBPb protein on both the C/EBPa and PPARg of GSK3b via the b-Catenin–Axin2 Cascade to Affect promoters (Fig. 5A–C). In parallel, expression of the Snail the Function of C/EBPb and Snail protein, but not mRNA, was either upregulated or down- GSK3b is a regulator that phosphorylates nuclear tran- regulated by activation or inhibition of Wnt/b-catenin scription factors such as C/EBPb and Snail to promote signaling in the above cells, respectively (Fig. 5D–F). adipogenesis (37–39). Similar to Axin1, Axin2 is a cyto- Finally, we assessed whether Wnt/b-catenin signaling plasmic protein and contains a homologous GSK3b binding and Pygo2 regulates adipogenesis via Axin2. This time, we domain (40). Therefore, we reasoned that Axin2 might knocked out b-catenin or Pygo2 by CRISPR/Cas9 tech- physically interact with GSK3b. To examine this possibil- nique with or without overexpression of Axin2 or Axin2- ity, we cotransfected Myc-tagged Axin2 or Axin2-DGSK3b DGSK3b to detect whether the above functions driven by and HA-tagged GSK3b to perform coimmunoprecipitation b-catenin or Pygo2 could be rescued by Axin2. The results experiments. As shown in Supplementary Fig. 1A, Axin2, showed that simultaneous overexpression of Axin2 signif- but not Axin2-DGSK3b, interacted with GSK3b. Next, we icantly compromised the b-catenin or Pygo2 knockout– examined whether Axin2 directly associates with GSK3b. induced increase in nuclear translocation of GSK3b, phos- An in vitro pulldown assay was performed, and it dem- phorylated C/EBPb (Fig. 6A and B), increase in C/EBPb onstrated direct binding between Axin2 and GSK3b (Sup- occupancy on the PPARg promoter (Fig. 6C and D), in- plementary Fig. 1B). Finally, we investigated whether the crease in adipogenesis (Fig. 6E), and decrease in Snail endogenous proteins interact by performing immunopre- proteinexpression(Fig.6A and B), whereas Axin2- cipitation experiments in 3T3-L1 cells. As shown in Sup- DGSK3b failed to exert this function. Similar to Axin2, plementary Fig. 1C, immunoprecipitation of GSK3b Axin1 is a cytoplasmic protein and contains a homolo- indeed pulled down Axin2. We conclude that there is gous GSK3b binding domain. We assessed whether Axin1 a direct interaction between Axin2 and GSK3b in preadi- exerts a similar function. Indeed, simultaneous overex- pocytes. This result implies that Axin2 may also keep pression of Axin1 to the level that could compensate for GSK3b in the cytoplasmic compartment by a retention the reduction of Axin2 also significantly compromised mechanism. To evaluate this model directly, we trans- the Pygo2 knockout–induced increase in phosphorylated fected Myc-tagged Axin2 or Axin2-DGSK3b in 3T3-L1 C/EBPb, increase in C/EBPb occupancy on the PPARg cells induced by MDI for 36 h, and GSK3b nuclear local- promoter, and decrease in Snail protein expression (Sup- ization was assessed. As shown in Fig. 4A, GSK3b localized plementary Fig. 2A and B). To further confirm that Pygo2 to both the cytosolic and nuclear compartments in con- inhibits adipogenesis through Axin2 in the opposite di- trol cells, and the introduction of Axin2 dramatically rection, Pygo2 was overexpressed and Axin2 was knocked reduced the nuclear distribution of GSK3b,whereasAxin2- down to the control level. As shown in Fig. 6F and G, Axin2 DGSK3b failed to exert this function. Next, we investi- knockdown could significantly restore the impaired adipo- gated whether Pygo2 regulates the cytoplasmic-nuclear genesis by Pygo2 overexpression. Together, these results diabetes.diabetesjournals.org Xie and Associates 2575

Figure 3—Pygo2 facilitates b-catenin signaling function in 3T3-L1 cells. A: Pygo2 enhances SuperTOPflash activity in the presence of b-catenin in preadipocytes. 3T3-L1 cells were transiently transfected with the indicated plasmids. SuperFOPflash where the Wnt-responsive elements are mutant was used as a control. B: Knockdown of Pygo2 expression using shRNAs in 3T3-L1 cells resulted in significantly reduced SuperTOPflash activity. Western blotting revealed the efficient knockdown of Pygo2 expression in 3T3-L1 cells. Overexpression (C) 2576 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018 functionally establish a relationship between Pygo2 and noted that Pygo2 expression was dramatically reduced in 2 2 the Wnt/b-catenin signaling–Axin2 cascade during adipo- PDGFRa+ adipocyte progenitors of Pygo2pre / animals, genesis. From these data, we propose the model for this which suggests that Pygo2 was effectively knocked out in mechanism as exhibited in Fig. 6H. these cells (Fig. 7C). We also assessed food intake, move- fi fi ment, and energy expenditure in control and knockout Pygo2-De cient Mice Show a Signi cant Increase pre2/2 in Adipogenesis and an Enlarged Fat Mass mice. On a normal chow diet, the Pygo2 mutation did To further investigate the role of Pygo2 in adipogenesis not affect food intake and movement (Supplementary Fig. in vivo, primary mouse embryonic fibroblasts (MEFs) were 4A and B). However, energy expenditure, including O2 2 2 / consumption, CO2 production, and heat production, was prepared from WT and Pygo2 mouse embryos at em- 2 2 fl lower in Pygo2pre / mice (Supplementary Fig. 4C–H). bryonic day 13.5, cultured to con uence, and then main- 2 2 Then, we monitored the weight of WT and Pygo2pre / tained for 3 weeks. The results showed that whereas WT 2 2/2 mice. No difference was found between WT and Pygo2pre / MEFs failed to differentiate into adipocytes, Pygo2 2 MEFs exhibited spontaneous adipocyte differentiation, mice at birth. On a normal chow diet, 14-week-old pre2/2 as verified by strong Oil Red O staining (Fig. 7A) and Pygo2 animals weighed more than their WT litter- an increase in C/EBPa and PPARg levels (Fig. 7B). We next mates (Fig. 7D). MRI examination showed increased ac- constructed a floxed allele of mouse Pygo2 and intro- cumulation of fat volume (Fig. 7E) and fat percentage (Fig. pre2/2 duced it into the PDGFRa-Cre transgene to generate the 7F)inPygo2 mice. These differences were also Pygo2 conditional knockout mice in adipocyte precur- mirrored by the increased weight of the epididymal fl fl pre2/2 sors (PDGFRa-Cre;Pygo2 ox/ ox) (41–43). The resulting WAT depot in Pygo2 mice (Fig. 7G). The increase fl fl PDGFRa-Cre;Pygo2 ox/ ox mice are henceforth referred to in adiposity was associated with a significant enlargement 2 2 pre2/2 as Pygo2pre / mice. Littermates lacking the Cre gene in adipocyte volume observed in Pygo2 mice com- fl fl (Pygo2 ox/ ox) were used as WT controls. It was reported pared with that in WT animals, as assessed by classic 2 that in addition to the Lin CD29+CD34+Sca-1+CD24+ hematoxylin-eosin staining (Fig. 7H and I). Moreover, adipocyte precursors, PDGFRa also labels small popula- this effect was accompanied by a significant increase in tions of CD45+CD31+ cells in WAT (43,44) and oligoden- the total content of genomic DNA in epididymal WAT, drocyte precursor cells (OPCs) in the brain (45). To exclude which suggests an increased number of adipocytes in pre2/2 the possibility that Pygo2 knockout may have effects on Pygo2 mice (Fig. 7J). To further examine whether pre2/2 immune cells in WAT or on the CNS, we assessed Pygo2 the increased adiposity in Pygo2 mice was associated 2 2 expression in these cell types in the WT and Pygo2pre / with changes in adipocyte differentiation, we analyzed mice. Real-time qPCR results indicated that Pygo2 was mature adipocyte–specific gene expression. The expression 2 expressed mainly in Lin CD29+CD34+Sca-1+CD24+ adipo- levels of C/EBPa,PPARg1, and PPARg2weresignifi- pre2/2 cyte precursors, very little in CD45+ and CD31+ cells, and cantly increased in the WAT of Pygo2 mice com- 2 2 was effectively deleted in Pygo2pre / mice (Supplemen- pared with those in WT mice. Moreover, the expression tary Fig. 7C). Moreover, when we isolated neurons and levels of PPARg target genes, including those coding for OPCs from mouse brain and checked the Pygo2 expression, lipogenic proteins, such as FABP4, fatty acid synthase real-time qPCR results showed that Pygo2 was highly (FASN), and fatty acid transport protein 1 (FATP1), as expressed in neurons but absent in OPCs (Supplemen- well as those coding for adipokines, such as adiponectin, tary Fig. 3A). Consistently, confocal immunofluorescence resistin, and leptin, were significantly increased in the 2 2 analysis of brain tissue revealed that Pygo2 was highly WAT of Pygo2pre / mice compared with those in WT mice expressed in NeuN+ neuron cells but absent in oligo2+ (Fig. 7K). Interestingly, the genes involved in fatty acid oligodendrocyte lineage cells (Supplementary Fig. 3B). oxidation in WAT, such as acyl-CoA dehydrogenase me- From these results, we conclude that Pygo2 was knocked dium chain (MCAD) and carnitine palmitoyltransferase 2 2 out only in adipocyte precursors in Pygo2pre / mice. 1b (Cpt1b), were significantly decreased in the WAT of 2 2 First, we checked the b-catenin signaling in adipocyte Pygo2pre / mice compared with those in WT mice (Fig. progenitors by sorting PDGFRa+ cells, and the real-time 7K), which may represent the cause of decreased energy 2 2 qPCR results indicated that Pygo2pre / animals exhibited expenditure in these mice. We also examined the adipose greatly reduced expression of Wnt target genes Axin2 and tissue inflammation status and brown adipose tissue (BAT) Lef1 than the WT littermates in these cells (Fig. 7C). It is phenotype in Pygo2 knockout mice by detecting the or knockdown (D) of Pygo2 in 3T3-L1 cells upregulates or downregulates the expression of the endogenous Wnt target gene Axin2 at both the mRNA and protein levels. The results of real-time qPCR (left) and Western blotting (right) are shown. E and F: 3T3-L1 cells were induced to differentiate for 7 days using either MDI or differentiation media lacking IBMX (DI). Oil Red O stain visualizes lipid drops. Scale bar, 50 mm(E). Western blotting analysis of expression levels of the indicated proteins (F). G and H: Pygo2 was knocked down and dnTCF4 was si- multaneously overexpressed in 3T3-L1 cells. After DI induced for 7 days, Oil Red O stain (scale bar, 50 mm) (G) and Western blotting (H) detected the degree of adipogenesis. For A–D, each bar represents the average value of three independent experiments and error bars represent SD. **P , 0.01; ***P , 0.001. Ctrl, control. diabetes.diabetesjournals.org Xie and Associates 2577

Figure 4—Pygo2 regulates the cytoplasmic-nuclear distribution and function of GSK3b. A: The introduction of Axin2, but not Axin2-DGSK3b, dramatically reduced the nuclear distribution of GSK3b. Myc-tagged Axin2 or Axin2-DGSK3b was transfected into 3T3-L1 cells induced by MDI for 36 h, and GSK3b nuclear localization was assessed by Western blotting. 3T3-L1 preadipocytes were infected with lentivirus carrying GFP or b-catenin (B), GFP or dnTCF4 (C), and control (Ctrl) shRNA or Pygo2 shRNA (D). The nuclear and cytoplasmic extracts were prepared after cells were induced with MDI for 36 h. The Axin2 level, the cytoplasmic-nuclear distribution of GSK3b, and the levels of other indicated proteins were assessed by Western blotting. Lamin B was used as a nuclear loading control, and GAPDH was used as a cytoplasmic loading control. E–G: 3T3-L1 cells were stably expressed with GFP or b-catenin (E), GFP or dnTCF4 (F), and Ctrl shRNA or Pygo2 shRNA (G). Whole- cell lysates were extracted after cells were induced by MDI for 36 h and treated with MG132 for 12 h. Western blotting revealed the phosphorylation of C/EBPb and Snail, the stability of Snail, and the expression levels of the other indicated proteins.

2 2 2 expression of TNFa and IL-6, as well as the histology of Pygo2pre / mice were achieved when the Pygo2+/ mice interscapular BAT. The results showed significantly in- were treated under high-fat diet (HFD) feeding for 6 weeks 2 2 creased TNFa and IL-6 expression in Pygo2pre / mice (Supplementary Fig. 6). It is possible that Pygo2 deletion (Fig. 7K), but no significant difference in the BAT pheno- prevents preadipocyte proliferation and/or commitment, 2 2 type between Pygo2pre / mice and WT controls (Supple- thereby limiting the number of committed preadipocytes. 2 mentary Fig. 5). Finally, we checked the above adipose To exclude this possibility, we sorted Lin CD29+CD34 2 tissue phenotypes in Pygo2+/ mice. When these adipose +Sca-1+CD24+ adipocyte precursors, and a similar popula- 2 2 2 tissue phenotypes were not changed in Pygo2+/ mice tion percentage was observed in both Pygo2pre / and comparedwiththoseinWTmiceundernormalchow control mice (Supplementary Fig. 7A and B). Real-time diet conditions, results similar to those observed in qPCR detection of proliferation markers, including Ki67 2578 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018

Figure 5—Pygo2 regulates the DNA binding activity of C/EBPb and the protein stability of Snail. 3T3-L1 cells were infected with lentivirus expressing GFP or b-catenin, GFP or dnTCF4, and control (Ctrl) shRNA or Pygo2 shRNA and induced by MDI for 36 h. A–C: ChIP assay indicated the occupancy of C/EBPb on C/EBPa and PPARg promoters, and the results were obtained by real-time qPCR. Western blotting assessed the expression of b-catenin (A), dnTCF4 (B), and Pygo2 (C). D–F: Snail protein, but not mRNA, expression was either upregulated or downregulated by activation or inhibition of Wnt signaling in 3T3-L1 cells, respectively. Snail mRNA levels were measured by real-time qPCR, and the indicated protein levels were detected by Western blotting. Each bar represents the average value of three independent experiments, and error bars represent SD. ***P , 0.001. and Pcna, exhibited similar expression levels (Supplemen- our in vivo observation that reveals an impaired glucose tary Fig. 7D). These results indicate that Pygo2 deletion tolerance and decreased systemic insulin sensitivity in does not affect preadipocyte commitment and/or prolif- Pygo2-deficient mice highlights the important role played eration. Collectively, the above data suggest that increased by Pygo2 in WAT and glucose homeostasis. adipogenesis and decreased energy expenditure may con- pre2/2 tribute to increased adiposity in Pygo2 mice. DISCUSSION Impaired Glucose Tolerance and Decreased Systemic Previous studies have established that Wnt/b-catenin Insulin Sensitivity in Pygo2-Deficient Mice signaling inhibits adipogenesis, and some Wnt/b-catenin Increased WAT mass is usually associated with impair- components are regulated in this process (19–22). Since ments in glucose metabolism and insulin sensitivity, lead- there are so many components within the Wnt/b-catenin ing to obesity (46–49). Therefore we examined the effect of signaling cascade, there must be other factors that can Pygo2 ablation on glucose homeostasis. The levels of blood govern the Wnt/b-catenin output level during adipogen- glucose and the concentration of fasting serum insulin esis. Indeed, our present study has identified Pygo2 as under normal chow diet conditions were similar in a newly identified activator of Wnt/b-catenin signaling in 2 2 Pygo2pre / and WT mice (Fig. 8A and B). However, during the regulation of adipocyte differentiation. We showed 2 2 a GTT, Pygo2pre / mice showed significantly higher blood that Pygo2 exhibited a declined expression pattern during glucose concentrations after glucose challenge (Fig. 8C). adipocyte differentiation, resulting in an attenuated Wnt/ Moreover, insulin secretion in response to glucose load b-catenin signaling output level. Ectopic expression of 2 2 also showed a delayed peak in Pygo2pre / mice (Fig. 8D). Pygo2 attenuates adipocyte differentiation, whereas The ITT was also conducted, which revealed that the knockdown of Pygo2 enhances the differentiation process glucose-lowering effect of insulin was more hampered in and induces spontaneous differentiation. Furthermore, the mutant mice than it was in WT mice (Fig. 8E). We also MEFs lacking Pygo2 exhibited spontaneous adipocyte 2 conducted the above experiments in Pygo2+/ mice. As differentiation, and adipocyte precursor–specific Pygo2 described in Supplementary Fig. 8, the metabolic param- deletion showed a significant increase in adipocyte differ- 2 eters were not changed in Pygo2+/ mice compared with entiation. Thus, our gain- and loss-of-function studies those in WT mice under normal chow diet conditions. both in vitro and in vivo have clearly indicated that 2 2 However, results similar to those in Pygo2pre / mice were Pygo2isanovelfactorrequiredfortheregulationof 2 achieved when the Pygo2+/ mice were treated under HFD Wnt/b-catenin signaling during adipocyte differentiation. feeding for 6 weeks (Supplementary Fig. 9). Collectively, It is noted in this study that Pygo2 was also examined to diabetes.diabetesjournals.org Xie and Associates 2579

Figure 6—Pygo2 regulates C/EBPb and Snail function via the b-catenin–Axin2–GSK3b cascade. A–E: The expression of b-catenin or Pygo2 was knocked out by the CRISPR/Cas9 technique with or without lentivirus overexpression of Axin2 or Axin2-DGSK3b. A and B: The nuclear translocation of GSK3b, phosphorylated C/EBPb, and Snail protein expression were detected by Western blotting. C and D: The C/EBPb occupancy on the PPARg promoter was assessed by ChIP assay. E: Adipocyte differentiation was visualized by Oil Red O staining. Scale bar, 50 mm. F and G: Pygo2 was overexpressed and Axin2 was simultaneously knocked down to the control level. The phosphorylated C/EBPb and Snail protein expression were detected by Western blotting (F). Adipocyte differentiation was visualized by Oil Red O staining. Scale bar, 50 mm(G). H: Schematic diagram summarizing the working model that indicates that Pygo2 inhibits adipogenesis via the b-catenin–Axin2– GSK3b cascade. The solid lines indicate the activated signaling events, whereas the dotted lines indicate the inhibited signaling events. For C and D, each bar represents the average value of three independent experiments and error bars represent SD. **P , 0.01; ***P , 0.001. Ctrl, control; KO, knockout. 2580 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018

Figure 7—Pygo2-deficient mice present increased adiposity and elevated gene expression related to adipogenesis and glucose metabolism. A and B: Primary MEFs were isolated from embryos of WT and Pygo22/2 mice at embryonic day 13.5, cultured to confluence, and then maintained for 3 weeks. Cells were stained with Oil Red O (A). Scale bar, 50 mm. Whole-cell lysates were prepared for Western blotting to measure Pygo2, C/EBPa, and PPARg levels (B). C: PDGFRa+ cells were sorted by FACS from adipose tissues, and real-time qPCR results indicated that Pygo2pre2/2 animals exhibited greatly reduced expression of Wnt target genes Axin2 and Lef1 than the WT littermates in PDGFRa+ cells. D: The body weight of 14-week-old male Pygo2pre2/2 and WT (n = 10) mice fed a normal chow diet was evaluated. E: The fat volume (right) and representative images (left) in 14-week-old Pygo2pre2/2 and WT mice fed a normal chow diet were acquired by MRI (n = 4). F: The ratio of fat to body weight in 14-week-old Pygo2pre2/2 and WT mice fed a normal chow diet (n = 4). G: The ratio of epididymal WAT (eWAT) to body weight in 14-week-old Pygo2pre2/2 and WT (n = 10) mice (left), and a macroscopic view of the eWAT (right). H: Hematoxylin- diabetes.diabetesjournals.org Xie and Associates 2581 express at a low level in the cytoplasm of 3T3-L1 cells overexpressed or knocked down Axin2 upon Wnt/b-cat- (Fig. 4D), although it is originally identified as a nuclear enin signaling inhibition or activation to the original level component of Wnt/b-catenin signaling. Whether this to rescue the related phenotypes. Nevertheless, we have protein is regulated to translocate between nucleus identified a novel mechanism for Axin2 in regulating and cytoplasm during adipogenesis is worthy of further adipogenesis. analysis. The most unexpected result of this study was that upon Wnt/b-catenin signaling has been demonstrated to PPARg upregulation, deletion of the Pygo2 gene in adipose block adipogenesis by suppressing the expression of tissue resulted in larger adipocytes as well as enhanced C/EBPa and PPARg, whereas the expression of C/EBPb insulin resistance and impaired glucose tolerance, which and C/EBPd was not affected (18,19). However, the un- seems to be opposite to the fundamental function of derlying molecular mechanism by which Wnt/b-catenin PPARg. Indeed, it has been generally accepted that acti- signaling drives this regulation is still incompletely de- vation of PPARg leads to amelioration of insulin re- fined. The main challenge to connect this gap is that we sistance, since administration of thiazolidinedione, the must identify known or unknown Wnt/b-catenin down- PPARg agonist, has been shown to increase insulin sen- stream effectors that also directly control the expression sitivity in obese insulin-resistant animals and humans of C/EBPa and PPARg. Previous studies identified COUP- (53,54). However, another study in heterozygous PPARg- TFII as a negative regulator of adipogenesis (50) and deficient mice showed that these mice were protected adirectWnt/b-catenin signaling downstream target from the development of insulin resistance due to adipo- that can directly inhibit PPARg1 and PPARg2 mRNA cyte hypertrophy under an HFD and they suggested expression (51). However, the function of COUP-TFII in that PPARg plays dual roles in the regulation of insulin adipogenesis is controversial, since another report of an sensitivity (55). Therefore, in some cases, activation of in vivo study showed that COUP-TFII inhibits the expres- PPARg ameliorates insulin resistance, whereas in other sion of Wnt10b and acts as a positive regulator of adipo- cases, the increase in the amount of the PPARg gene genesis (52). In our system, we did not find the activation product leads to insulin resistance. In our case, Pygo2 of COUP-TFII by either b-catenin or Pygo2 (Supplemen- deletion results in increased adipogenesis and decreased tary Fig. 10). Instead, we uncovered a new mechanism energy expenditure. Because the adipose expandability for acting upstream of C/EBPb to mediate this process (Fig. an individual is limited, the adipose tissue in Pygo2-de- 6H). We found that inhibition of Wnt/b-catenin signaling letion mice tends to differentiate to mature adipocyte but by overexpressing dnTCF4 or knocking down b-catenin or finally will reach the maximal fixed capacity for safely Pygo2 resulted in the downregulation of Axin2, a consti- storing fat. During the adolescent period of mice, the tutive Wnt target, in the cytoplasm. Consequently, the increase in differentiated adipocytes still allows the storage Axin2-bound GSK3b was released and translocated into of an increasing amount of fat. In adult mice, once the the nucleus to phosphorylate C/EBPb and Snail. C/EBPb is accumulation of fat is beyond the speed of adipocyte the main upstream activator of C/EBPa and PPARg (18), production, PPARg activation results in adipocyte hyper- and Snail has been demonstrated to be a negative regulator trophy to generate large adipocytes. Once the adipocyte of adipogenesis by inhibiting PPARg expression (39). size is enlarged to the limit of its expandability, the excess Moreover, phosphorylation of C/EBPb by GSK3b results lipids will overflow from the adipose tissue. Consequently, in an increase in DNA binding activity (37), whereas TNFa and IL-6 release from large adipocytes and should be phosphorylation of Snail by GSK3b decreases its protein the cause of glucose intolerance and insulin resistance. stability (38). Therefore, by this complex signal cascade, Although we have identified the decreased expression of Pygo2 or b-catenin controls adipocyte differentiation. The fatty acid oxidation genes in Pygo2-deletion mice as the original article on Wnt inhibition of adipogenesis showed main cause of decreased energy expenditure, the under- that overexpression of Axin promotes adipogenesis (18), lying detailed mechanism needs to be further investigated which seems to be inconsistent with our data. We reasoned in the future. that the dosage and the context are all different. First, in As discussed above, obesity has been implicated in an overexpression study, people usually overexpress the glucose intolerance and insulin resistance (56,57). Indeed, objective protein to hundreds of times of the endogenous we found a decreased expression level of Pygo2 in ob/ob level. In our study, Axin2 is only overexpressed or knocked mice in comparison with WT mice. When we deleted Pygo2 down within physiological dosage. Second, because Axin2 expression in mouse adipocyte precursors, an enlarged is a downstream target of Wnt/b-catenin signaling, we fat mass was observed even under normal chow diet

eosin staining of the eWAT of 14-week-old Pygo2pre2/2 and WT mice. Scale bar, 50 mm. I: Quantification of the adipocyte size in the eWAT in 14-week-old Pygo2pre2/2 and WT mice (n = 4). J: Total DNA content of eWAT in Pygo2pre2/2 (n = 5) and WT (n = 6) mice. K: Real-time qPCR analysis of the indicated mRNAs in the eWAT of Pygo2pre2/2 and WT mice (n = 3) (bottom). Pygo2 levels in mouse SVF were detected by Western blotting (top). For C–G and I–K, error bars represent SEM. *P , 0.05; **P , 0.01; ***P , 0.001. 2582 Pygo2 Regulates Adiposity and Blood Glucose Diabetes Volume 67, December 2018

Figure 8—Impaired glucose tolerance and decreased systemic insulin sensitivity in Pygo2-deficient mice. Feeding and fasting blood glucose levels (A) and fasting serum insulin concentration (B) were examined in 14-week-old Pygo2pre2/2 (n = 8) and WT mice (n = 7) fed a normal chow diet. C and D: GTT. The blood glucose (C) and serum insulin (D) levels in 16-week-old Pygo2pre2/2 (n = 8) and WT (n = 6) mice fed a normal chow diet were measured at the indicated times after the intraperitoneal injection of 2 g/kg body weight of glucose. The graphs represent the area under the curve (AUC) (C, right). E: ITT. The blood glucose levels in 15-week-old Pygo2pre2/2 and WT mice fed a normal chow diet were measured at the indicated times after the intraperitoneal injection of 0.75 units/kg body weight of insulin (n = 7). The graphs represent the AUC. Error bars represent SEM. *P , 0.05. We found no evidence for deviation of the data points from a normal distribution, or for differences in variance between groups of mice under analysis.

conditions, which suggests an obesity tendency. Both GTT study also revealed an association between Pygo2 function and ITT assays also showed abnormal blood glucose and and obesity or diabetes. insulin concentrations in Pygo2-deletion mice. It is noted that the glucose and insulin levels were synchronously regulated in WT mice, which exhibited the same peak Funding. This work was supported by grants from the National Natural Science Foundation of China (grant numbers U1705284, 81472457, and 81772958 to value occurrence time at 15 min after glucose challenge. B.-A.L. and 81472725 to W.M.) and “Project 111” sponsored by the State However, the insulin peak value occurrence time of Pygo2- Bureau of Foreign Experts and Ministry of Education (grant number B06016). deletion mice was delayed to 30 min after glucose chal- Duality of Interest. No potential conflicts of interest relevant to this article lenge, which suggests impaired glucose-stimulated insulin were reported. secretion, and therefore perhaps a defect in b-cell func- Author Contributions. Y.-Y.X. conceived the study, designed and per- tion. Interestingly, the delayed insulin peak value in Pygo2- formed experiments, analyzed data, and wrote the manuscript. C.-L.M., Y.-H.C., deletion mice was increased. We deduced that this might W.-J.W., K.-K.Z., and W.M. performed experiments and analyzed data. X.-X.H., be the compensation for the functional abnormality of in- Q.-F.L., Y.-J.L., J.-J.H., T.H., and Z.-Z.Z. constructed various plasmids for the sulin in the Pygo2-deletion obese mice. Collectively, these study. B.-A.L. conceived the study, designed experiments, and wrote the man- data have linked the two important roles played by Pygo2 uscript. B.-A.L. is the guarantor of this work and, as such, had full access to all the in adipose metabolism and glucose metabolism. data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. In summary, we have defined a new effector that determines the Wnt/b-catenin output level during adipo- References genesis. Moreover, we identified a novel molecular signal 1. Spiegelman BM, Flier JS. Obesity and the regulation of energy balance. Cell cascade that mediates Wnt/b-catenin signaling activity in 2001;104:531–543 adipose tissues, which established an association between 2. Scherer PE. Adipose tissue: from lipid storage compartment to endocrine C/EBPa/PPARg expression and Wnt/b-catenin activation. organ. Diabetes 2006;55:1537–1545 Our study highlighted the role of Wnt/b-catenin in the 3. Must A, Spadano J, Coakley EH, Field AE, Colditz G, Dietz WH. The disease regulation of adipogenesis and glucose homeostasis. This burden associated with overweight and obesity. JAMA 1999;282:1523–1529 diabetes.diabetesjournals.org Xie and Associates 2583

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