Diabetes Volume 66, March 2017 689

Bin Liu,1 Han Lu,2 Duanzhuo Li,1 Xuelian Xiong,3 Lu Gao,4,5 Zhixiang Wu,6 and Yan Lu1,3

Aberrant Expression of FBXO2 Disrupts Glucose Homeostasis Through -Mediated Degradation of Insulin Receptor in Obese Mice

Diabetes 2017;66:689–698 | DOI: 10.2337/db16-1104

Insulin resistance is a critical factor in the development of secretion deficiency and/or reduced insulin sensitivity. In metabolic disorders, including type 2 diabetes (T2DM). peripheral tissues, including liver, skeletal muscle, and However, its molecular mechanisms remain incompletely adipose tissue, insulin binds to its receptor (IR), which then understood. In this study, we found that F-box only phosphorylates and recruits IR substrates (IRSs) to further 2 (FBXO2), a substrate recognition component of the activate downstream signaling pathways (1). In the liver, BST STUDIES OBESITY Skp1-Cul1-F-box protein (SCF) E3 com- the major node of insulin signaling is activation of phos- plex, was upregulated in livers of obese mice. Further- phoinositide-3-kinase/AKT, which in turn inhibits the ex- fi more, using a protein puri cation approach combined pression of phosphoenolpyruvate carboxykinase (PEPCK) with high-performance liquid chromatography/tandem and glucose-6-phosphatase (G6Pase), two key gluconeo- mass spectrometry, we carried out a system-wide screen- genic enzymes (2). As a result, hepatic insulin resistance ing of FBXO2 substrates, in which the insulin receptor (IR) is characterized by excessive hepatic glucose production, was identified as a substrate for FBXO2. SCFFBXO2 acts as contributing to fasting hyperglycemia in T2DM (3). There- an E3 ligase targeting the IR for ubiquitin-dependent deg- fi radation to regulate insulin signaling integrity. As a result, fore, identi cation of novel molecules involved in regulat- adenovirus-mediated overexpression of FBXO2 in healthy ing the hepatic insulin signaling pathway will advance our mice led to hyperglycemia, glucose intolerance, and insu- understanding of the pathogenesis that leads to T2DM. lin resistance, whereas ablation of FBXO2 alleviated dia- Polyubiquitination is the formation of an ubiquitin betic phenotypes in obese mice. Therefore, our results chain on a single lysine residue on the substrate protein, identify SCFFBXO2 as an E3 ligase for the IR in the liver, leading to protein degradation (4). It is carried out by a three- which might provide a novel therapeutic target for treating step cascade of ubiquitin transfer reactions—activation, con- T2DM and related metabolic disorders. jugation, and ligation—performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively (5). The largest Type 2 diabetes (T2DM), characterized by high blood subfamily of E3s in mammals is the Skp1-Cul1-F-box glucose concentrations, has become a pandemic problem protein ubiquitin ligases, which consist of Skp1, Cul1, worldwide. Hyperglycemia is usually caused by an insulin Rbx1, and one of the F-box (FBPs) (6). Recent

1Hubei Key Laboratory for Kidney Disease Pathogenesis and Intervention, Corresponding authors: Lu Gao, [email protected], Zhixiang Wu, zhixiangwu@ Huangshi Cental Hospital of Edong Healthcare Group, Hubei Polytechnic Univer- yahoo.com, and Yan Lu, [email protected]. sity School of Medicine, Huangshi, Hubei, China Received 8 September 2016 and accepted 1 December 2016. 2Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao Tong University This article contains Supplementary Data online at http://diabetes School of Medicine, Shanghai, China .diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1104/-/DC1. 3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan Uni- versity, Shanghai, China B.L. and H.L. are co–first authors. 4College of Life Sciences, Northeast Agricultural University, Harbin, Heilongjiang, © 2017 by the American Diabetes Association. Readers may use this article as China long as the work is properly cited, the use is educational and not for profit, and the 5Department of Pathology, University of Maryland School of Medicine, Baltimore, work is not altered. More information is available at http://www.diabetesjournals MD .org/content/license. 6Department of Pediatric Surgery, Xinhua Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai, China 690 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017 studies have shown that FBPs play a crucial role in many a 100-mm inner diameter, packed with 5 mm C18 resin) at a biological events, such as inflammation, cell cycle progres- flow rate of 5 mL/min in 100% buffer A (0.1% formic acid sion, and tumorigenesis, through ubiquitin-mediated degra- in high-performance liquid chromatography–grade water). dation of cellular regulatory proteins (7,8). In addition, their After 10 min of loading and washing, the peptides were trans- dysregulation has been implicated in several pathologies ferred to an analytical column (17 cm 3 79 mm, 3-mmpar- (6–8), suggesting that insights into Skp1-Cul1-F-box protein ticle size; Dikma Co, Beijing, China) coupled to an Easy nLC ubiquitin ligase–mediated biology may provide potential 1000 system (Thermo Fisher Scientific). The separated pep- strategies to treat human diseases. Until now, however, tides were ionized using a nanospray ionization source, then whether FBPs play a role in metabolic diseases, especially analyzed in an Orbitrap Fusion mass spectrometer (Thermo insulin resistance and T2DM, remains poorly understood. Fisher Scientific) with a top speed 3s data-dependent mode. For MS/MS scanning, ions with an intensity above 5,000 and RESEARCH DESIGN AND METHODS charge states 2–6 in each full MS spectrum were sequentially Animal Experiments fragmented by higher collision dissociation, with normalized Male C57BL/6 and db/db mice aged 8–10 weeks were pur- collision energy of 32%. The dynamic exclusion duration was chased from the Shanghai Laboratory Animal Company set at 60 s, and the precursor ions were isolated by quadru- and Nanjing Biomedical Research Institute of Nanjing Uni- pole with a 1-Da isolation window. The fragment ions were versity, respectively. JNK1 knockout mice were obtained analyzed in the ion trap with automatic gain control 7,000 at from The Jackson Laboratory and backcrossed to a rapid scan mode. The raw spectra data were processed by C57BL/6 background for six generations. All mice were Thermo Proteome Discoverer 2.1 and MS/MS spectra data housed at 216 1°C with humidity of 55% 6 10% and a were searched against the Uniprot human database (88,817 12-h light/12-h dark cycle. Mice with high-fat diet (HFD)– sequences) by Mascot (v.2.4; Matrix Science, London, U.K.). induced obesity were maintained with free access to high- Bioinformatics Analysis fat chow (D12492; Research Diets, Inc) containing 60% The molecular function and cellular components of the kcal from fat, 20% kcal from carbohydrate, and 20% kcal glycoproteins were analyzed using the Database for Annota- from protein. For the depletion of Kupffer cells, C57BL/6 tion, Visualization and Integrated Discovery Bioinformatics mice were fed an HFD for 12 weeks and then injected with Database (DAVID 6.7) (10,11). gadolinium chloride (GdCl3; 10 mg/kg, twice each week) or sodium chloride (NaCl) by tail vein for another 2 weeks. Glucose and Insulin Tolerance Tests All study protocols comply with guidelines and institu- Glucose tolerance tests (GTTs) were performed by intraper- tional policies prepared by the Animal Care Committee itoneal injection of D-glucose (Sigma-Aldrich) at a dose of of Shanghai Jiao Tong University School of Medicine. 2.0 mg/g body weight after a 16-h fast. For insulin tolerance tests (ITTs), mice were injected with regular human insulin Immuoprecipitation and In-Solution Digestion (Eli Lilly & Company, Indianapolis, IN) at a dose of 0.75 fi The standard immunoprecipitation (IP) puri cation pro- U/kg body weight after a 6-h fast. Blood glucose was mea- cedure has been previously described (9). In brief, HEK293T sured using a portable blood glucose meter (LifeScan; cells stably expressing Flag-tagged wild-type (WT) or mutant Johnson & Johnson, New Brunswick, NJ). (MUT) F-box only protein 2 (FBXO2) were lysed in 5 mL lysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/L Western Blotting NaCl, 0.5% Nonidet P40, and 100 mmol/L phenylmethylsul- Hepatic tissues or cells were lysed in radioimmunoprecipi- fonyl fluoride) with gentle rocking at 4°C for 20 min. Lysates tation buffer containing 50 mmol/L Tris-HCl, 150 mmol/L were cleared and subjected to IP with 50 mL of anti-FLAG NaCl, 5 mmol/L MgCl2, 2 mmol/L EDTA, 1 mmol/L NaF, M2 beads overnight at 4°C. Beads containing immune com- 1% NP-40, and 0.1% SDS. Western blotting was performed plexes were washed with 1 mL ice-cold lysis buffer. Proteins using antibodies against FBXO2 (ab133717; Abcam), IRb were eluted with 100 mL 3X FLAG peptide (Sigma-Aldrich, (ab131238; Abcam), AKT (13038, 4821; Cell Signaling Tech- St. Louis, MO) in Tris-buffered saline for 30 min and pre- nologies), and GAPDH (5174; Cell Signaling Technologies). cipitated with cold acetone. The precipitated proteins were Tyrosine phosphorylation of IRS1 was analyzed by IP of digested in solution with trypsin, and the tryptic peptides IRS1 with anti-IRS1 from total lysate, followed by Western were centrifuged in a vacuum to dryness for further analysis. blotting with anti-pTyr antibody (PY100). High-Performance Liquid Chromatography/Tandem Luciferase Reporter and Chromatin IP Assays Mass Spectrometry Analysis All the transient transfections were conducted using Lip- Nanoflow liquid chromatography/tandem mass spectrome- ofectamine 2000 (Invitrogen, Shanghai, China). The FBXO2 try (MS) was performed by coupling an Easy nLC 1000 liquid promoter was amplified from the mouse genomic DNA chromatograph (Thermo Fisher Scientific, Waltham, MA) to templates and inserted into pGL4.15 empty vector (Prom- an Orbitrap Fusion mass spectrometer (Thermo Fisher ega). Luciferase activity was measured using the Dual- Scientific). Tryptic peptides were dissolved in 20 mLof Luciferase Reporter Assay System (Promega). For chromatin 0.1% formic acid, and 10 mL were injected for each analysis. IP (ChIP) assays, a commercial kit was used (Upstate, Peptides were delivered to a trap column (2-cm length with Billerica, MA). In short, mouse primary hepatocytes (MPHs) diabetes.diabetesjournals.org Liu and Associates 691 were fixedwithformaldehyde,andchromatinwasincubated in HFD-fed mice was further confirmed by quantitative and precipitated with antibodies against p65 (ab16502; real-time PCR and Western blotting, respectively (Fig. 1A Abcam) or control IgG (ab172730; Abcam). DNA fragments and B). Upregulation of FBXO2 was also detected in the were subjected to real-time PCR using primers flanking the livers of db/db mice (Fig. 1C and D), a well-established nuclear factor (NF)-kB binding site in the FBXO2 promoter. genetic model of T2DM, suggesting that abnormal expres- The primer sequences were 59-ACCAGCGCGACGCGG TAT sion of FBXO2 represents a typical feature of insulin re- GGGA-39 (forward) and 59-TGGGGCAGCCGGACTAAAA sistance in obese animals. GCT-39 (reverse). Identification of the IR as a Novel Substrate for FBXO2 Statistical Analysis FBXO2 was shown to preferentially target N-linked high- Values are shown as mean 6 SEM. Statistical differences mannose oligosaccharides in glycoproteins for ubiquitina- were determined using the Student t test. Statistical signif- tion and degradation (14). The F-box–associated domain icance is considered at P , 0.05, P , 0.01, or P , 0.001. of FBXO2 is essential for its activity of recognizing gly- coprotein, which is completely abolished by mutations RESULTS of two residues (15,16). To systematically identify Upregulation of FBXO2 in Livers of Obese Mice the FBXO2-interacting proteins, HEK293T cells were To identify that are differentially expressed in obesity, transfected with retroviruses expressing Flag-tagged WT we previously performed a clustering analysis of Affymetrix FBXO2 or an F-box–associated domain mutant (MUT), arrays, which showed that a large number of mRNAs were which could not recognize glycoprotein, as previously de- markedly dysregulated in the liver of mice fed an HFD scribed (15,16). IP against Flag was subsequently per- compared with mice fed a normal chow diet (12,13). Here formed with the lysates of cells carrying WT or MUT we describe work on the FBPs. More than 70 FBPs are FBXO2 proteins, respectively. As depicted in Supplemen- present in mammals (6). Our data showed that 11 FBPs tary Fig. 2, all purification procedures were monitored were significantly changed (P , 0.05), of which 8 were in- by Coomassie Brilliant Blue staining as well as Western creased and 3 were decreased (Supplementary Table 1). blotting with anti-Flag antibody, showing that both WT Here, FBXO2 was chosen for further experiments because and MUT FBXO2 proteins were highly enriched in the its expression was enriched in the liver and hepatocytes final elution fraction. Consistent with previous results (16), (Supplementary Fig. 1A and B). By contrast, its expression concanavalin positivity signals accumulated dramatically in in other tissues, including skeletal muscle, white adipose the WT, but not MUT, final elution fraction. The final tissue, heart, and kidney, was relatively low (Supplementary immunoprecipitates from WT and MUT cells were further Fig. 1A). Increased mRNA and protein expression of FBXO2 subjected to MS analysis. Proteins were identified using

Figure 1—FBXO2 expression in the liver. Relative mRNA (A) and representative protein levels (B) of FBXO2, determined by quantitative real-time PCR and Western blotting, in livers of C57BL/6 mice. Eight-week-old mice were fed a normal chow diet (ND) or an HFD for 12 weeks (n = 6). Hepatic mRNA (C) and protein levels (D) of FBXO2 in db/db mice (n = 8). ***P < 0.001. IB, immunoblotting. 692 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017

Mascot software, and identified proteins filtered with an glycoproteins were highly enriched in membrane, endoplas- overall false discovery rate ,0.01% were considered as po- mic reticulum (ER), and lysosome (Fig. 2C and Supplemen- tential interacting candidates. Using these criteria, we finally taryTable3).GiventherelevanceofFBXO2inobeseanimals, identified 2,643 proteins from WT samples and 1,138 pro- we questioned whether any molecules involved in the insulin teins from MUT samples (Supplementary Table 2). To ex- signaling pathway are potential substrates of FBXO2. Intrigu- clude the unspecificbinding,wethenfocusedontheproteins ingly, we found that IR, a large transmembrane glycoprotein that were exclusively identified in WT cells, resulting in 1,569 containing multiple N-linked glycosylation sites (17,18), was potential substrates. Importantly, through comparison with coeluted with only WT FBXO2, and not MUT FBXO2, in two the Uniprot database, we found that more than one-third of replicates (Fig. 2D). these proteins (528, or 33.7%) were glycoproteins. By con- trast, only 82 proteins (7.6%) from MUT elutes were classi- FBXO2 Negatively Regulates the Stability of the IR fied as glycoproteins in the Uniprot database (Fig. 2A). Next, we confirmed the specific interaction between FBXO2 Together, our data indicated that glycoproteins were signifi- and the IR in transiently transfected HEK293T cells using cantly enriched among the proteins interacted with WT but coimmunoprecipitation (Fig. 3A). The endogenous interac- not MUT FBXO2. Interestingly, the Kyoto Encyclopedia of tion of these two proteins was also detected in MPHs (Fig. Genes and Genomes pathway showed that a portion of these 3B). Because FBXO2 could interact with the IR, we tested glycoproteins was involved in N-glycan biosynthesis and whether FBXO2 could regulate IR stability or accelerate its oxidative phosphorylation, suggesting a potential role for protein degradation. Indeed, endogenous IR protein contents FBXO2 in energy metabolism (Fig. 2B and Supplementary were dramatically decreased in MPHs transfected with ade- Table 3). Bioinformatics analysis further showed that these novirus expressing FBXO2 (Fig. 3C), whereas its mRNA levels

Figure 2—Identification of IR as a novel interacting protein for FBXO2. A: Venn diagram of the proteins identified from WT and MUT FBXO2 interacting proteins. B: Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of the glycoproteins exclusively identified from cells over- expressing WT FBXO2. ECM, extracellular matrix; GPI, glycophosphatidylinositol. C: ontology analysis of the glycoproteins exclusively identified from cells overexpressing WT FBXO2. D: Spectra counting–based quantification analysis of IR protein from WT and MUT FBXO2 interacting proteins. R1 and R2 represent two replicates. diabetes.diabetesjournals.org Liu and Associates 693

Figure 3—FBXO2 negatively regulates the stability of IR. A: Western blots of coimmunoprecipitated FBXO2 from HEK293T cells trans- fected with Flag-tagged FBXO2 and hemagglutinin (HA)-tagged IR. Cells were pretreated with MG132 for 4 h. B: FBXO2 was immumo- precipitated from MPHs using anti-FBXO2 or IgG antibody. Whole-cell extracts and IPs were separated by SDS-PAGE and immunoblotted for the proteins indicated. C: Endogenous expression of IR, IRS1, IRS2, Glut1, Glut4, and IGF1R proteins were determined in MPHs overexpressing FBXO2 or green fluorescent protein (GFP) for 48 h. D: Relative mRNA level of IR in MPHs. E: IR ubiquitination in MPHs overexpressing FBXO2 or GFP. Cells were pretreated with MG132 for 4 h. Ub, ubiquitin. F: Time course of IR levels in cycloheximide (CHX)- treated MPHs with or without FBXO2 overexpression (left); quantification is shown on the right. IB, immunoblotting.

remained unchanged (Fig. 3D). In addition, abundance of E), indicating the involvement of the proteasome system in IRS1, IRS2, Glut1, and Glut4 proteins were not affected by FBXO2-mediated inhibition of insulin signaling. FBXO2 overexpression (Fig. 3C). The IGF-I receptor, which is closely related to the IR and has overlapping functions, was Liver-Specific Overexpression of FBXO2 Promotes slightly reduced, suggesting the specificity of FBXO2-induced Hyperglycemia and Insulin Resistance IR degradation (Fig. 3C). The ubiquitination of IR was also To investigate the role of FBXO2 in regulating insulin increased by ectopic expression of FBXO2 in MPHs treated signaling in vivo, FBXO2 or green fluorescent protein with MG132, a proteasome inhibitor (Fig. 3E). Furthermore, adenovirus was administered to C57BL/6 mice via a tail overexpression of FBXO2 reduced the half-life of IR to less vein injection. As shown in Fig. 5A, the level of FBXO2 than 2 h (Fig. 3F), supporting the notion that FBXO2 could protein was dramatically increased while IR was decreased regulate IR stability and promote its degradation. In agree- in the liver, but not in other tissues, including white ment, posttranscriptional downregulation of hepatic IR was adipose tissues and skeletal muscles (data not shown). also observed in obese mice (Supplementary Fig. 3A–D). Overexpression of hepatic FBXO2 did not affect body Moreover, insulin inhibited dexamethasone/foskolin- weight or food intake (Supplementary Fig. 4A and B), but induced glucose production, which was largely attenuated by significantly increased circulating concentrations of glu- the overexpression of FBXO2 (Fig. 4A). In agreement with cose and insulin, indicating insulin resistance (Fig. 5B this, FBXO2 expression also blocked the suppressive effects and C). A dramatic reduction in insulin sensitivity was of insulin on dexamethasone/foskolin-induced expression also revealed by GTTs and ITTs (Fig. 5D). These changes of gluconeogenic enzymes (PEPCK and G6Pase) (Fig. 4B). were accompanied at a molecular level by phosphorylation In addition, FBXO2-induced downregulation of IR protein of IRS1 and AKT, two crucial molecules in the insulin was attenuated by MG132 (a proteasome inhibitor), but not signaling pathway, in response to acute intraperitoneal leupeptin (an inhibitor of lysosomal protease) (Fig. 4C). insulin injection (Fig. 5E). Moreover, the mRNA expres- MG132 treatment also restored insulin-suppressed glucose sion of PEPCK and G6Pase was upregulated by FBXO2 production and gluconeogenic (Fig. 4D and overexpression (Fig. 5F). 694 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017

Figure 4—The inhibitory effects of insulin on glucose production and gluconeogenic gene expression are blocked by FBXO2 overexpres- sion. A and B: Glucose production (A) and gene expression (B) in MPHs overexpressing FBXO2 or green fluorescent protein (GFP). The effects of insulin on cAMP/dexamethasone (DEX)-induced glucose production were measured with a colorimetric glucose assay kit. The mRNA expression of PEPCK and G6Pase was quantified by real-time PCR. C: Representative protein levels of IR and FBXO2 in MPHs overexpressing FBXO2 or GFP. Cells were treated with MG132 or leupeptin for 6 h before harvest. D and E: Relative glucose production (D) and gene expression (E) in MPHs. Cells were treated with MG132 for 6 h before harvest. **P < 0.01; ***P < 0.001. FSK, foskolin; IB, immunoblotting; n.s, not significant.

Ablation of FBXO2 Enhances Insulin Sensitivity in with FBXO2 shRNA (Supplementary Fig. 5A–D), suggesting db/db Mice that knockdown of FBXO2 in the liver could alleviate the To further confirm the effects of FBXO2inanindependent diabetic phenotype in obese mice. setting, we disrupted its expression in the liver of db/db mice by delivering adenovirus-expressing FBXO2-specificshort Regulation of Hepatic FBXO2 in Obesity hairpin RNA (shRNA) or a nonspecific control shRNA. The results described above demonstrate that FBXO2 was FBXO2 shRNA treatment significantly reduced hepatic upregulated in obese livers, and manipulation of FBXO2 FBXO2 protein levels and increased IR protein expression could modulate insulin sensitivity. Finally, we sought compared with negative control shRNA-injected littermates to determine the signaling pathway that regulates FBXO2 (Fig. 6A). As a result, loss of FBXO2 dramatically improved expression. T2DM is tightly associated with high circulating hyperglycemia, hyperinsulinemia, glucose tolerance, and in- concentrations of glucose, fatty acids, insulin, and proin- sulin resistance (Fig. 6B–D). Well-improved insulin signaling flammatory cytokines. Therefore we performed a screen to and downregulation of gluconeogenic enzymes were also assess whether these cellular factors and hormones could observed in db/db mice with FBXO2 deficiency (Fig. 6E affect FBXO2 expression. As a result, tumor necrosis factor-a and F). Similar effects on glucose homeostasis were observed (TNF-a) and interleukin (IL)-1b, but not high glucose, fatty in mice with HFD-induced obesity that were transduced acids, insulin, or dexamethasone, induced FBXO2 expression diabetes.diabetesjournals.org Liu and Associates 695

Figure 5—Overexpression of FBXO2 impairs the hepatic actions of insulin and induces hyperglycemia in C57BL/6 mice. A: Representative Western blots showing levels of FBXO2 protein in the liver of C57BL/6 mice at day 14 after infection with adenoviruses encoding FBXO2 or green fluorescent protein (GFP) control. B–D: Blood glucose (B) and insulin (C) concentrations and GTTs and ITTs (D) in C57BL/6 mice. Data were obtained on day 5 (B and C), day 8 (D, GTT), and day 11 (D, ITT) after virus administration. For insulin concentrations, 30-mL aliquots of blood were collected at 9:00 A.M. from individual mice (n =8).E: Phosphorylation of IRS1 and AKT in response to acute insulin injection in C57BL/6 mice. Mice were fasted overnight and injected intraperitoneally with insulin (0.75 U insulin/kg body weight) or saline. After injection (10 min), liver tissues were harvested for homogenization. F: Relative mRNA levels of PEPCK and G6Pase from two groups of mice (n =8).**P < 0.01; ***P < 0.001. IB, immunoblotting.

in MPHs (Fig. 7A and Supplementary Fig. 6A–D), suggesting BAY 11–7082 (an NF-kB inhibitor), but not SP600125 (a that inflammation might be responsible for the upregulation JNK inhibitor) or U0126 (an extracellular signal–regulated of FBXO2 in obese mice. To confirm this point, we deleted kinase inhibitor) (Supplementary Fig. 7B), suggesting that Kupffer cells in HFD-fed mice by administering GdCl3 (19). the canonical IKKb/NF-kB pathway mediates the effects of Consistent with previous reports that Kupffer cells are the proinflammatory cytokines to induce FBXO2 expression. primary source for hepatic inflammation in obesity (19–21), Next, we speculated that FBXO2 is a molecular target of fi b k fi GdCl3 treatment signi cantly reduced the expression of pro- IKK /NF- B. To con rm this, we examined the promoter inflammatory markers including TNF-a,IL-1b, and F4/80 in region of FBXO2 and found that a canonical NF-kBDNA- liver tissues (Supplementary Fig. 6E). Under this condition, binding motif (59-GGGRNNYYCC-39) exists in the proximal there was a marked decrease in FBXO2 expression in the promoter region of the FBXO2 gene (Fig. 7C). We then livers of obese mice compared with controls (Fig. 7B). created luciferase plasmids controlled by the FBXO2 Growing evidence has noted the roles of inflammation- promoter and found that IKKb increased the transcrip- mediated c-Jun N-terminal kinase (JNK) 1 and IKKb/NF-kB tional activities of these promoters when transfected into signaling pathways on the regulation of liver metabolic ho- HEK293T cells (Fig. 7D). On the other hand, mutagenesis meostasis (20,22–24). Hence, it is interesting to determine of the NF-kB DNA-binding motif abrogated the effect of whether JNK1 and/or IKKb/NF-kB activation may underlie IKKb/NF-kB in activating the transcriptional activities of the upregulation of FBXO2. As shown in Supplementary Fig. these promoters (Fig. 7D). Similarly, inhibition of NF-kB 7A, FBXO2 mRNA expression showed similar changes after activation by BAY 11–7082 abolished the TNF-a–induced TNF-a treatment in JNK1 knockout MPHs compared with activity of the FBXO2 promoter (Fig. 7E), further suggest- JNK1 WT MPHs, suggesting that JNK1 might not be essen- ing that hepatic inflammation regulates FBXO2 through tial for the regulation of FBXO2 expression. In agreement NF-kB signaling. The association of p65 with the FBXO2 with this, the induction of FBXO2 was largely blocked by promoter was also confirmed by ChIP assays (Fig. 7F). 696 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017

Figure 6—Knockdown of FBXO2 alleviates the diabetic phenotype in db/db obese mice. A: Quantitative real-time PCR and Western blot analysis to detect the mRNA and protein levels of IR and FBXO2 in the liver of db/db mice at day 15 after infection with adenoviral FBXO2 shRNA or LacZ shRNA (n = 8 or 9). B–D: Blood glucose (B) and insulin (C) concentrations and GTTs and ITTs (D)indb/db mice. Data were obtained on day 5 (B and C), day 8 (D, GTT), and day 12 (D, ITT) after virus administration (n = 8 or 9). E: Phosphorylation of IRS1 and AKT in response to acute insulin injection in db/db mice. Mice were fasted overnight and injected intraperitoneally with insulin (0.75 U insulin/kg body weight) or saline for 10 min. F: Relative mRNA levels of PEPCK and G6Pase from two groups of db/db mice (n =8or9).**P < 0.01; ***P < 0.001. IB, immunoblotting.

Considering these data together, we speculate that chronic upregulated in obese livers, suggesting that inhibiting the hepatic inflammation–mediated IKKb/NF-kBactivation expression or activity of FBXO2 might represent a potential may be an important mechanism leading to upregulation therapeutic target for enhancing insulin sensitivity. of FBXO2 in obesity. Several studies since the 1970s have reported the abnormal number and function of IRs in various tissues DISCUSSION of insulin-resistant mice, including liver, adipose tissue, Previous studies have created mice, via the Cre-loxP system, skeletal muscle, leukocytes, and endothelial cells, whereas with tissue-specific disruption of the IR gene. Intriguingly, its mRNA levels were not found to be decreased (28–31). hyperglycemia and insulin resistancewereonlyexhibitedin These results suggest that the small number of receptors liver-specific IR knockout mice and not in skeletal muscle– could be due to posttranscriptional levels. Indeed, it has or fat-specificIRknockoutmice(25–27), suggesting that been shown that IR protein expression could be targeted hepatic IR has a critical role in regulating glucose homeo- and inhibited by several microRNAs in adipocytes, heart, stasis and insulin sensitivity. Although downstream signal- and liver (32–34). Song et al. (35) demonstrated that IR is ing pathways of insulin are well established, molecular ubiquitinated by Mitsugumin 53 (MG53) in skeletal muscle determinants that directly regulate IR expression remain because IR ubiquitination and insulin-elicited downstream poorly elucidated. In this study we provide in vitro and signaling are inversely changed in MG53 transgenic mice in vivo evidence showing a critical role of FBXO2 as a post- and MG53 knockout mice. A recent study identified nu- transcriptional regulator of hepatic insulin signaling. First, a clear ubiquitous casein and cyclin-dependent kinase sub- protein purification approach combined with the high- strate as regulators of IR expression, thereby regulating performance liquid chromatography/tandem MS assay was energy homeostasis and glucose metabolism (36). There- used to identify IR as a novel interacting protein of FBXO2, fore, along with these studies, molecular interventions that which was further confirmed by coimmunoprecipitation selectively increase IR expression might provide an attrac- assays. FBXO2 interacts with the IR to enhance its tive avenue to treat T2DM. Although both we and another ubiquitination-mediated protein degradation. Second, the group (35) found that proteasome inhibitor administration physiological role of FBXO2 is further revealed by both could efficiently prevent the degradation of IR by different gain-of-function and loss-of-function studies of mice. Over- E3 ligases, how IR gets into the proteasome for degra- expression of FBXO2 in the liver led to hyperglycemia, dation remains unclear. Moreover, our bioinformatics hyperinsulinemia, glucose intolerance, and insulin resistance analyses showed that the glycoproteins that interacted in healthy mice, whereas selective knockdown of FBXO2 in exclusively with WT FBXO2 were highly enriched in the obese mice improved these symptoms. Third, FBXO2 was membrane, ER, and lysosome, suggesting other membrane diabetes.diabetesjournals.org Liu and Associates 697

Figure 7—Regulation of FBXO2 by activation of the IKKb/NF-kB pathway. A: Relative mRNA levels of FBXO2 in MPHs treated with TNF-a (10 ng/mL) or IL-1b (10 ng/mL) for the indicated time. B: Relative mRNA and representative protein levels of FBXO2 in HFD-fed mice. Mice were fed an HFD for 12 weeks and then treated with GdCl3 or NaCl for another 2 weeks (n = 6). C: Proximal promoter region of the mouse FBXO2 gene contains a potential binding site for NF-kB. D and E: Luciferase reporter assays. HEK293T cells were transfected with luciferase reporter plasmids containing WT or MUT binding site of NF-kB. Cells were treated with vehicle control (DMSO) or BAY 11– 7082, an inhibitor of NF-kB activation. F: ChIP assays showing representative p65 binding to the FBXO2 promoter in MPHs. Cells were treated with TNF-a or PBS for 2 h and then subjected to ChIP assays. *P < 0.05; **P < 0.01; ***P < 0.001. EV, empty vector; IB, immunoblotting. glycoproteins might also be ubiquitinated by FBXO2. Mem- novel insight whereby inflammation inhibits the hepatic brane proteins are subject to a complex series of sorting, actions of insulin. In addition,whetherFBXO2expression trafficking, quality control, and quality maintenance sys- could be regulated by other factors such as ER stress and tems, which are largely controlled by ubiquitination (37). autophagy remains to be determined. Retrotranslocation of misfolded membrane proteins from To our knowledge, we have for the first time identified the ER into the cytoplasm and processive cleavage by the FBXO2 as a functional E3 ligase for IR in the liver. Several 26S proteasome also participate in ubiquitination-mediated recent reports showed that FBXO2 plays an important role degradation (38). Interestingly, it has been reported that in the brain by controlling the abundance of the N-methyl- FBXO2 ubiquitinates N-glycosylated proteins that are D-aspartate receptor and amyloid precursor protein (40,41). translocated from the ER to the cytosol and functions However, its role in other biological events remains in an ER-associated degradation pathway (14). Therefore, largely unexplored. Therefore, future studies directed the degradation of IR might take place in the ER via retro- at understanding its tissue-specific downstream targets translocation, which needs to be determined in future studies. are needed. Our data also indicate that aberrant expression of FBXO2 is attributed, at least in part, to the activation of IKKb/NF- k fl Bbyproinammatory factors. Numerous studies have Acknowledgments. The authors are grateful to Xiaoying Li from fl demonstrated that low-grade and chronic in ammation Zhongshan Hospital, Fudan University, Shanghai, for helpful discussion of plays a positive role in the glucose intolerance and insulin the manuscript. resistance seen in obesity (39). While several potential mech- Funding. This study was supported by grants from the National Natural anisms have been proposed (39), our results may provide a Science Foundation of China (grant nos. 81402478, 31401185, and 81570769), the 698 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017

Shanghai Rising-Star Program (grant no. 16QA1402900), and the Research 20. Lackey DE, Olefsky JM. Regulation of metabolism by the innate immune Foundation of Hubei Polytechnic University for Talented Scholars (grant no. 9666). system. Nat Rev Endocrinol 2016;12:15–28 Duality of Interest. No potential conflicts of interest relevant to this article 21. Stienstra R, Saudale F, Duval C, et al. Kupffer cells promote hepatic were reported. steatosis via interleukin-1beta-dependent suppression of peroxisome proliferator- Author Contributions. B.L., H.L., and L.G. performed animal and cellular activated receptor alpha activity. Hepatology 2010;51:511–522 experiments and analyzed the data. D.L. and X.X. provided technical advice on 22. Pal M, Febbraio MA, Lancaster GI. The roles of c-Jun NH2-terminal kinases the cellular studies. Z.W. and Y.L. conceived the research ideas, supervised the (JNKs) in obesity and insulin resistance. J Physiol 2016;594:267–279 project, and wrote the manuscript. Y.L. is the guarantor of this work and, as 23. Manieri E, Sabio G. Stress kinases in the modulation of metabolism and such, had full access to all the data in the study and takes responsibility for the energy balance. J Mol Endocrinol 2015;55:R11–R22 integrity of the data and the accuracy of the data analysis. 24. Cai D, Yuan M, Frantz DF, et al. Local and systemic insulin resistance resulting from hepatic activation of IKK-beta and NF-kappaB. Nat Med 2005;11:183–190 References 25. Blüher M, Michael MD, Peroni OD, et al. Adipose tissue selective insulin 1. Bornfeldt KE, Tabas I. Insulin resistance, hyperglycemia, and atheroscle- receptor knockout protects against obesity and obesity-related glucose in- rosis. Cell Metab 2011;14:575–585 tolerance. Dev Cell 2002;3:25–38 2. Pernicova I, Korbonits M. Metformin–mode of action and clinical implica- 26. Michael MD, Kulkarni RN, Postic C, et al. Loss of insulin signaling in he- tions for diabetes and cancer. Nat Rev Endocrinol 2014;10:143–156 patocytes leads to severe insulin resistance and progressive hepatic dysfunction. 3. Könner AC, Brüning JC. Selective insulin and leptin resistance in metabolic Mol Cell 2000;6:87–97 disorders. Cell Metab 2012;16:144–152 27. Brüning JC, Michael MD, Winnay JN, et al. A muscle-specific insulin re- 4. Hoeller D, Dikic I. Targeting the ubiquitin system in cancer therapy. Nature ceptor knockout exhibits features of the metabolic syndrome of NIDDM without 2009;458:438–444 altering glucose tolerance. Mol Cell 1998;2:559–569 5. Vucic D, Dixit VM, Wertz IE. Ubiquitylation in apoptosis: a post-translational 28. Du J, Fan LM, Mai A, Li JM. Crucial roles of Nox2-derived oxidative stress in modification at the edge of life and death. Nat Rev Mol Cell Biol 2011;12:439–452 deteriorating the function of insulin receptors and endothelium in dietary obesity 6. Skaar JR, Pagan JK, Pagano M. SCF ubiquitin ligase-targeted therapies. Nat of middle-aged mice. Br J Pharmacol 2013;170:1064–1077 Rev Drug Discov 2014;13:889–903 29. Ludwig S, Muller-Wieland D, Goldstein BJ, Kahn CR. The insulin receptor 7. Zheng N, Wang Z, Wei W. Ubiquitination-mediated degradation of cell cycle- gene and its expression in insulin-resistant mice. Endocrinology 1988;123:594– related proteins by F-box proteins. Int J Biochem Cell Biol 2016;73:99–110 600 8. Wang Z, Liu P, Inuzuka H, Wei W. Roles of F-box proteins in cancer. Nat Rev 30. Soli AH, Kahn CR, Neville DM Jr, Roth J. Insulin receptor deficiency in Cancer 2014;14:233–247 genetic and acquired obesity. J Clin Invest 1975;56:769–780 9. Liu B, Zheng Y, Wang TD, et al. Proteomic identification of common SCF 31. Kahn CR, Neville DM Jr, Roth J. Insulin-receptor interaction in the obese- ubiquitin ligase FBXO6-interacting glycoproteins in three kinds of cells. J Pro- hyperglycemic mouse. A model of insulin resistance. J Biol Chem 1973;248: teome Res 2012;11:1773–1781 244–250 10. Huang DW, Sherman BT, Tan Q, et al. DAVID Bioinformatics Resources: 32. Wu D, Xi QY, Cheng X, et al. miR-146a-5p inhibits TNF-a-induced adipo- expanded annotation database and novel algorithms to better extract biology from genesis via targeting insulin receptor in primary porcine adipocytes. J Lipid Res large gene lists. Nucleic Acids Res 2007;35(Web server issue):W169–W175 2016;57:1360–1372 11. Huang W, Sherman BT, Lempicki RA. Systematic and integrative analysis of 33. Marchand A, Atassi F, Mougenot N, et al. miR-322 regulates insulin sig- large gene lists using DAVID bioinformatics resources. Nat Protoc 2009;4:44–57 naling pathway and protects against metabolic syndrome-induced cardiac dys- 12. Lu Y, Ma Z, Zhang Z, et al. Yin Yang 1 promotes hepatic steatosis through function in mice. Biochim Biophys Acta 2016;1862:611–621 repression of farnesoid X receptor in obese mice. Gut 2014;63:170–178 34. Wang X, Wang M, Li H, et al. Upregulation of miR-497 induces hepatic 13. Lu Y, Liu X, Jiao Y, et al. Periostin promotes liver steatosis and hyper- insulin resistance in E3 rats with HFD-MetS by targeting insulin receptor. Mol Cell triglyceridemia through downregulation of PPARa. J Clin Invest 2014;124:3501–3513 Endocrinol 2015;416:57–69 14. Yoshida Y, Chiba T, Tokunaga F, et al. E3 ubiquitin ligase that recognizes 35. Song R, Peng W, Zhang Y, et al. Central role of E3 ubiquitin ligase MG53 in sugar chains. Nature 2002;418:438–442 insulin resistance and metabolic disorders. Nature 2013;494:375–379 15. Mizushima T, Yoshida Y, Kumanomidou T, et al. Structural basis for the 36. Qiu B, Shi X, Wong ET, et al. NUCKS is a positive transcriptional regulator of selection of glycosylated substrates by SCF(Fbs1) ubiquitin ligase. Proc Natl Acad insulin signaling. Cell Reports 2014;7:1876–1886 Sci U S A 2007;104:5777–5781 37. MacGurn JA, Hsu PC, Emr SD. Ubiquitin and membrane protein turnover: 16. Glenn KA, Nelson RF, Wen HM, Mallinger AJ, Paulson HL. Diversity in tissue from cradle to grave. Annu Rev Biochem 2012;81:231–259 expression, substrate binding, and SCF complex formation for a lectin family of 38. Okiyoneda T, Apaja PM, Lukacs GL. Protein quality control at the plasma ubiquitin ligases. J Biol Chem 2008;283:12717–12729 membrane. Curr Opin Cell Biol 2011;23:483–491 17. Sparrow LG, Lawrence MC, Gorman JJ, et al. N-linked glycans of the hu- 39. Schenk S, Saberi M, Olefsky JM. Insulin sensitivity: modulation by nutrients man insulin receptor and their distribution over the crystal structure. Proteins and inflammation. J Clin Invest 2008;118:2992–3002 2008;71:426–439 40. Atkin G, Moore S, Lu Y, et al. Loss of F-box only protein 2 (Fbxo2) disrupts 18. Elleman TC, Frenkel MJ, Hoyne PA, et al. Mutational analysis of the N-linked levels and localization of select NMDA receptor subunits, and promotes aberrant glycosylation sites of the human insulin receptor. Biochem J 2000;347:771–779 synaptic connectivity. J Neurosci 2015;35:6165–6178 19. Tomiyama K, Ikeda A, Ueki S, et al. Inhibition of Kupffer cell-mediated early 41. Atkin G, Hunt J, Minakawa E, et al. F-box only protein 2 (Fbxo2) regulates proinflammatory response with carbon monoxide in transplant-induced hepatic amyloid precursor protein levels and processing. J Biol Chem 2014;289:7038– ischemia/reperfusion injury in rats. Hepatology 2008;48:1608–1620 7048