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

Aberrant Expression of FBXO2DisruptsGlucose Homeostasis ThroughUbiquitin-Mediated Degradationof Insulin Receptor in Obese MiceDiabetes 2017;66:689–698 | DOI: 10.2337/db16-1104

Insulin resistance is a critical factor in the development ofmetabolic disorders, including type 2 diabetes (T2DM).However, its molecular mechanisms remain incompletelyunderstood. In this study, we found that F-box only protein2 (FBXO2), a substrate recognition component of theSkp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase com-plex, was upregulated in livers of obese mice. Further-more, using a protein purification approach combinedwith high-performance liquid chromatography/tandemmass spectrometry, we carried out a system-wide screen-ing of FBXO2 substrates, in which the insulin receptor (IR)was identified as a substrate for FBXO2. SCFFBXO2 acts asan E3 ligase targeting the IR for ubiquitin-dependent deg-radation to regulate insulin signaling integrity. As a result,adenovirus-mediated overexpression of FBXO2 in healthymice led to hyperglycemia, glucose intolerance, and insu-lin resistance, whereas ablation of FBXO2 alleviated dia-betic phenotypes in obese mice. Therefore, our resultsidentify SCFFBXO2 as an E3 ligase for the IR in the liver,which might provide a novel therapeutic target for treatingT2DM and related metabolic disorders.

Type 2 diabetes (T2DM), characterized by high bloodglucose concentrations, has become a pandemic problemworldwide. Hyperglycemia is usually caused by an insulin

secretion deficiency and/or reduced insulin sensitivity. Inperipheral tissues, including liver, skeletal muscle, andadipose tissue, insulin binds to its receptor (IR), which thenphosphorylates and recruits IR substrates (IRSs) to furtheractivate downstream signaling pathways (1). In the liver,the major node of insulin signaling is activation of phos-phoinositide-3-kinase/AKT, which in turn inhibits the ex-pression of phosphoenolpyruvate carboxykinase (PEPCK)and glucose-6-phosphatase (G6Pase), two key gluconeo-genic enzymes (2). As a result, hepatic insulin resistanceis characterized by excessive hepatic glucose production,contributing to fasting hyperglycemia in T2DM (3). There-fore, identification of novel molecules involved in regulat-ing the hepatic insulin signaling pathway will advance ourunderstanding of the pathogenesis that leads to T2DM.

Polyubiquitination is the formation of an ubiquitinchain on a single lysine residue on the substrate protein,leading to protein degradation (4). It is carried out by a three-step cascade of ubiquitin transfer reactions—activation, con-jugation, and ligation—performed by ubiquitin-activatingenzymes (E1s), ubiquitin-conjugating enzymes (E2s),and ubiquitin ligases (E3s), respectively (5). The largestsubfamily of E3s in mammals is the Skp1-Cul1-F-boxprotein ubiquitin ligases, which consist of Skp1, Cul1,Rbx1, and one of the F-box proteins (FBPs) (6). Recent

1Hubei Key Laboratory for Kidney Disease Pathogenesis and Intervention,Huangshi Cental Hospital of Edong Healthcare Group, Hubei Polytechnic Univer-sity School of Medicine, Huangshi, Hubei, China2Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao Tong UniversitySchool of Medicine, Shanghai, China3Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan Uni-versity, Shanghai, China4College of Life Sciences, Northeast Agricultural University, Harbin, Heilongjiang,China5Department of Pathology, University of Maryland School of Medicine, Baltimore,MD6Department of Pediatric Surgery, Xinhua Hospital, Shanghai Jiao Tong UniversitySchool of Medicine, Shanghai, China

Corresponding authors: Lu Gao, gaolu@neau.edu.cn, Zhixiang Wu, zhixiangwu@yahoo.com, and Yan Lu, lu.yan2@zs-hospital.sh.cn.

Received 8 September 2016 and accepted 1 December 2016.

This article contains Supplementary Data online at http://diabetes.diabetesjournals.org/lookup/suppl/doi:10.2337/db16-1104/-/DC1.

B.L. and H.L. are co–first authors.

© 2017 by the American Diabetes Association. Readers may use this article aslong as the work is properly cited, the use is educational and not for profit, and thework is not altered. More information is available at http://www.diabetesjournals.org/content/license.

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OBESITYSTUDIES

studies have shown that FBPs play a crucial role in manybiological events, such as inflammation, cell cycle progres-sion, and tumorigenesis, through ubiquitin-mediated degra-dation of cellular regulatory proteins (7,8). In addition, theirdysregulation has been implicated in several pathologies(6–8), suggesting that insights into Skp1-Cul1-F-box proteinubiquitin ligase–mediated biology may provide potentialstrategies to treat human diseases. Until now, however,whether FBPs play a role in metabolic diseases, especiallyinsulin resistance and T2DM, remains poorly understood.

RESEARCH DESIGN AND METHODS

Animal ExperimentsMale C57BL/6 and db/db mice aged 8–10 weeks were pur-chased from the Shanghai Laboratory Animal Companyand Nanjing Biomedical Research Institute of Nanjing Uni-versity, respectively. JNK1 knockout mice were obtainedfrom The Jackson Laboratory and backcrossed to aC57BL/6 background for six generations. All mice werehoused at 216 1°C with humidity of 55% 6 10% and a12-h light/12-h dark cycle. Mice with high-fat diet (HFD)–induced obesity were maintained with free access to high-fat chow (D12492; Research Diets, Inc) containing 60%kcal from fat, 20% kcal from carbohydrate, and 20% kcalfrom protein. For the depletion of Kupffer cells, C57BL/6mice were fed an HFD for 12 weeks and then injected withgadolinium chloride (GdCl3; 10 mg/kg, twice each week) orsodium chloride (NaCl) by tail vein for another 2 weeks.All study protocols comply with guidelines and institu-tional policies prepared by the Animal Care Committeeof Shanghai Jiao Tong University School of Medicine.

Immuoprecipitation and In-Solution DigestionThe standard immunoprecipitation (IP) purification pro-cedure has been previously described (9). In brief, HEK293Tcells stably expressing Flag-tagged wild-type (WT) or mutant(MUT) F-box only protein 2 (FBXO2) were lysed in 5 mLlysis buffer (50 mmol/L Tris-HCl [pH 7.5], 150 mmol/LNaCl, 0.5% Nonidet P40, and 100 mmol/L phenylmethylsul-fonyl fluoride) with gentle rocking at 4°C for 20 min. Lysateswere cleared and subjected to IP with 50 mL of anti-FLAGM2 beads overnight at 4°C. Beads containing immune com-plexes were washed with 1 mL ice-cold lysis buffer. Proteinswere eluted with 100 mL 3X FLAG peptide (Sigma-Aldrich,St. Louis, MO) in Tris-buffered saline for 30 min and pre-cipitated with cold acetone. The precipitated proteins weredigested in solution with trypsin, and the tryptic peptideswere centrifuged in a vacuum to dryness for further analysis.

High-Performance Liquid Chromatography/TandemMass Spectrometry AnalysisNanoflow liquid chromatography/tandem mass spectrome-try (MS) was performed by coupling an Easy nLC 1000 liquidchromatograph (Thermo Fisher Scientific, Waltham, MA) toan Orbitrap Fusion mass spectrometer (Thermo FisherScientific). Tryptic peptides were dissolved in 20 mL of0.1% formic acid, and 10 mL were injected for each analysis.Peptides were delivered to a trap column (2-cm length with

a 100-mm inner diameter, packed with 5 mm C18 resin) at aflow rate of 5 mL/min in 100% buffer A (0.1% formic acidin high-performance liquid chromatography–grade water).After 10 min of loading and washing, the peptides were trans-ferred to an analytical column (17 cm 3 79 mm, 3-mm par-ticle size; Dikma Co, Beijing, China) coupled to an Easy nLC1000 system (Thermo Fisher Scientific). The separated pep-tides were ionized using a nanospray ionization source, thenanalyzed in an Orbitrap Fusion mass spectrometer (ThermoFisher Scientific) with a top speed 3s data-dependent mode.For MS/MS scanning, ions with an intensity above 5,000 andcharge states 2–6 in each full MS spectrum were sequentiallyfragmented by higher collision dissociation, with normalizedcollision energy of 32%. The dynamic exclusion duration wasset at 60 s, and the precursor ions were isolated by quadru-pole with a 1-Da isolation window. The fragment ions wereanalyzed in the ion trap with automatic gain control 7,000 atrapid scan mode. The raw spectra data were processed byThermo Proteome Discoverer 2.1 and MS/MS spectra datawere searched against the Uniprot human database (88,817sequences) by Mascot (v.2.4; Matrix Science, London, U.K.).

Bioinformatics AnalysisThe molecular function and cellular components of theglycoproteins were analyzed using the Database for Annota-tion, Visualization and Integrated Discovery BioinformaticsDatabase (DAVID 6.7) (10,11).

Glucose and Insulin Tolerance TestsGlucose tolerance tests (GTTs) were performed by intraper-itoneal injection of D-glucose (Sigma-Aldrich) at a dose of2.0 mg/g body weight after a 16-h fast. For insulin tolerancetests (ITTs), mice were injected with regular human insulin(Eli Lilly & Company, Indianapolis, IN) at a dose of 0.75U/kg body weight after a 6-h fast. Blood glucose was mea-sured using a portable blood glucose meter (LifeScan;Johnson & Johnson, New Brunswick, NJ).

Western BlottingHepatic tissues or cells were lysed in radioimmunoprecipi-tation buffer containing 50 mmol/L Tris-HCl, 150 mmol/LNaCl, 5 mmol/L MgCl2, 2 mmol/L EDTA, 1 mmol/L NaF,1% NP-40, and 0.1% SDS. Western blotting was performedusing antibodies against FBXO2 (ab133717; Abcam), IRb(ab131238; Abcam), AKT (13038, 4821; Cell Signaling Tech-nologies), and GAPDH (5174; Cell Signaling Technologies).Tyrosine phosphorylation of IRS1 was analyzed by IP ofIRS1 with anti-IRS1 from total lysate, followed by Westernblotting with anti-pTyr antibody (PY100).

Luciferase Reporter and Chromatin IP AssaysAll the transient transfections were conducted using Lip-ofectamine 2000 (Invitrogen, Shanghai, China). The FBXO2promoter was amplified from the mouse genomic DNAtemplates and inserted into pGL4.15 empty vector (Prom-ega). Luciferase activity was measured using the Dual-Luciferase Reporter Assay System (Promega). For chromatinIP (ChIP) assays, a commercial kit was used (Upstate,Billerica, MA). In short, mouse primary hepatocytes (MPHs)

690 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017

were fixed with formaldehyde, and chromatin was incubatedand precipitated with antibodies against p65 (ab16502;Abcam) or control IgG (ab172730; Abcam). DNA fragmentswere subjected to real-time PCR using primers flanking thenuclear factor (NF)-kB binding site in the FBXO2 promoter.The primer sequences were 59-ACCAGCGCGACGCGG TATGGGA-39 (forward) and 59-TGGGGCAGCCGGACTAAAAGCT-39 (reverse).

Statistical AnalysisValues are shown as mean 6 SEM. Statistical differenceswere determined using the Student t test. Statistical signif-icance is considered at P , 0.05, P , 0.01, or P , 0.001.

RESULTS

Upregulation of FBXO2 in Livers of Obese MiceTo identify genes that are differentially expressed in obesity,we previously performed a clustering analysis of Affymetrixarrays, which showed that a large number of mRNAs weremarkedly dysregulated in the liver of mice fed an HFDcompared with mice fed a normal chow diet (12,13). Herewe describe work on the FBPs. More than 70 FBPs arepresent in mammals (6). Our data showed that 11 FBPswere significantly changed (P , 0.05), of which 8 were in-creased and 3 were decreased (Supplementary Table 1).Here, FBXO2 was chosen for further experiments becauseits expression was enriched in the liver and hepatocytes(Supplementary Fig. 1A and B). By contrast, its expressionin other tissues, including skeletal muscle, white adiposetissue, heart, and kidney, was relatively low (SupplementaryFig. 1A). Increased mRNA and protein expression of FBXO2

in HFD-fed mice was further confirmed by quantitativereal-time PCR and Western blotting, respectively (Fig. 1Aand B). Upregulation of FBXO2 was also detected in thelivers of db/db mice (Fig. 1C and D), a well-establishedgenetic model of T2DM, suggesting that abnormal expres-sion of FBXO2 represents a typical feature of insulin re-sistance in obese animals.

Identification of the IR as a Novel Substrate for FBXO2FBXO2 was shown to preferentially target N-linked high-mannose oligosaccharides in glycoproteins for ubiquitina-tion and degradation (14). The F-box–associated domainof FBXO2 is essential for its activity of recognizing gly-coprotein, which is completely abolished by mutationsof two residues (15,16). To systematically identifythe FBXO2-interacting proteins, HEK293T cells weretransfected with retroviruses expressing Flag-tagged WTFBXO2 or an F-box–associated domain mutant (MUT),which could not recognize glycoprotein, as previously de-scribed (15,16). IP against Flag was subsequently per-formed with the lysates of cells carrying WT or MUTFBXO2 proteins, respectively. As depicted in Supplemen-tary Fig. 2, all purification procedures were monitoredby Coomassie Brilliant Blue staining as well as Westernblotting with anti-Flag antibody, showing that both WTand MUT FBXO2 proteins were highly enriched in thefinal elution fraction. Consistent with previous results (16),concanavalin positivity signals accumulated dramatically inthe WT, but not MUT, final elution fraction. The finalimmunoprecipitates from WT and MUT cells were furthersubjected 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 quantitativereal-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 for12 weeks (n = 6). Hepatic mRNA (C) and protein levels (D) of FBXO2 in db/db mice (n = 8). ***P < 0.001. IB, immunoblotting.

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Mascot software, and identified proteins filtered with anoverall false discovery rate ,0.01% were considered as po-tential interacting candidates. Using these criteria, we finallyidentified 2,643 proteins from WT samples and 1,138 pro-teins from MUT samples (Supplementary Table 2). To ex-clude the unspecific binding, we then focused on the proteinsthat were exclusively identified in WT cells, resulting in 1,569potential substrates. Importantly, through comparison withthe Uniprot database, we found that more than one-third ofthese proteins (528, or 33.7%) were glycoproteins. By con-trast, only 82 proteins (7.6%) from MUT elutes were classi-fied as glycoproteins in the Uniprot database (Fig. 2A).Together, our data indicated that glycoproteins were signifi-cantly enriched among the proteins interacted with WT butnot MUT FBXO2. Interestingly, the Kyoto Encyclopedia ofGenes and Genomes pathway showed that a portion of theseglycoproteins was involved in N-glycan biosynthesis andoxidative phosphorylation, suggesting a potential role forFBXO2 in energy metabolism (Fig. 2B and SupplementaryTable 3). Bioinformatics analysis further showed that these

glycoproteins were highly enriched in membrane, endoplas-mic reticulum (ER), and lysosome (Fig. 2C and Supplemen-tary Table 3). Given the relevance of FBXO2 in obese animals,we questioned whether any molecules involved in the insulinsignaling pathway are potential substrates of FBXO2. Intrigu-ingly, we found that IR, a large transmembrane glycoproteincontaining multiple N-linked glycosylation sites (17,18), wascoeluted with only WT FBXO2, and not MUT FBXO2, in tworeplicates (Fig. 2D).

FBXO2 Negatively Regulates the Stability of the IRNext, we confirmed the specific interaction between FBXO2and the IR in transiently transfected HEK293T cells usingcoimmunoprecipitation (Fig. 3A). The endogenous interac-tion of these two proteins was also detected in MPHs (Fig.3B). Because FBXO2 could interact with the IR, we testedwhether FBXO2 could regulate IR stability or accelerate itsprotein degradation. Indeed, endogenous IR protein contentswere dramatically decreased in MPHs transfected with ade-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 FBXO2interacting 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: Gene ontology analysis of the glycoproteins exclusivelyidentified from cells overexpressing WT FBXO2. D: Spectra counting–based quantification analysis of IR protein from WT and MUT FBXO2interacting proteins. R1 and R2 represent two replicates.

692 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017

remained unchanged (Fig. 3D). In addition, abundance ofIRS1, IRS2, Glut1, and Glut4 proteins were not affected byFBXO2 overexpression (Fig. 3C). The IGF-I receptor, which isclosely related to the IR and has overlapping functions, wasslightly reduced, suggesting the specificity of FBXO2-inducedIR degradation (Fig. 3C). The ubiquitination of IR was alsoincreased by ectopic expression of FBXO2 in MPHs treatedwith MG132, a proteasome inhibitor (Fig. 3E). Furthermore,overexpression of FBXO2 reduced the half-life of IR to lessthan 2 h (Fig. 3F), supporting the notion that FBXO2 couldregulate IR stability and promote its degradation. In agree-ment, posttranscriptional downregulation of hepatic IR wasalso observed in obese mice (Supplementary Fig. 3A–D).

Moreover, insulin inhibited dexamethasone/foskolin-induced glucose production, which was largely attenuated bythe overexpression of FBXO2 (Fig. 4A). In agreement withthis, FBXO2 expression also blocked the suppressive effectsof insulin on dexamethasone/foskolin-induced expressionof gluconeogenic enzymes (PEPCK and G6Pase) (Fig. 4B).In addition, FBXO2-induced downregulation of IR proteinwas attenuated by MG132 (a proteasome inhibitor), but notleupeptin (an inhibitor of lysosomal protease) (Fig. 4C).MG132 treatment also restored insulin-suppressed glucoseproduction and gluconeogenic gene expression (Fig. 4D and

E), indicating the involvement of the proteasome system inFBXO2-mediated inhibition of insulin signaling.

Liver-Specific Overexpression of FBXO2 PromotesHyperglycemia and Insulin ResistanceTo investigate the role of FBXO2 in regulating insulinsignaling in vivo, FBXO2 or green fluorescent proteinadenovirus was administered to C57BL/6 mice via a tailvein injection. As shown in Fig. 5A, the level of FBXO2protein was dramatically increased while IR was decreasedin the liver, but not in other tissues, including whiteadipose tissues and skeletal muscles (data not shown).Overexpression of hepatic FBXO2 did not affect bodyweight or food intake (Supplementary Fig. 4A and B), butsignificantly increased circulating concentrations of glu-cose and insulin, indicating insulin resistance (Fig. 5Band C). A dramatic reduction in insulin sensitivity wasalso revealed by GTTs and ITTs (Fig. 5D). These changeswere accompanied at a molecular level by phosphorylationof IRS1 and AKT, two crucial molecules in the insulinsignaling pathway, in response to acute intraperitonealinsulin injection (Fig. 5E). Moreover, the mRNA expres-sion of PEPCK and G6Pase was upregulated by FBXO2overexpression (Fig. 5F).

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 immunoblottedfor the proteins indicated. C: Endogenous expression of IR, IRS1, IRS2, Glut1, Glut4, and IGF1R proteins were determined in MPHsoverexpressing FBXO2 or green fluorescent protein (GFP) for 48 h. D: Relative mRNA level of IR in MPHs. E: IR ubiquitination in MPHsoverexpressing 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.

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Ablation of FBXO2 Enhances Insulin Sensitivity indb/db MiceTo further confirm the effects of FBXO2 in an independentsetting, we disrupted its expression in the liver of db/dbmiceby delivering adenovirus-expressing FBXO2-specific shorthairpin RNA (shRNA) or a nonspecific control shRNA.FBXO2 shRNA treatment significantly reduced hepaticFBXO2 protein levels and increased IR protein expressioncompared with negative control shRNA-injected littermates(Fig. 6A). As a result, loss of FBXO2 dramatically improvedhyperglycemia, hyperinsulinemia, glucose tolerance, and in-sulin resistance (Fig. 6B–D). Well-improved insulin signalingand downregulation of gluconeogenic enzymes were alsoobserved in db/db mice with FBXO2 deficiency (Fig. 6Eand F). Similar effects on glucose homeostasis were observedin mice with HFD-induced obesity that were transduced

with FBXO2 shRNA (Supplementary Fig. 5A–D), suggestingthat knockdown of FBXO2 in the liver could alleviate thediabetic phenotype in obese mice.

Regulation of Hepatic FBXO2 in ObesityThe results described above demonstrate that FBXO2 wasupregulated in obese livers, and manipulation of FBXO2could modulate insulin sensitivity. Finally, we soughtto determine the signaling pathway that regulates FBXO2expression. T2DM is tightly associated with high circulatingconcentrations of glucose, fatty acids, insulin, and proin-flammatory cytokines. Therefore we performed a screen toassess whether these cellular factors and hormones couldaffect FBXO2 expression. As a result, tumor necrosis factor-a(TNF-a) and interleukin (IL)-1b, but not high glucose, fattyacids, insulin, or dexamethasone, induced FBXO2 expression

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). Theeffects of insulin on cAMP/dexamethasone (DEX)-induced glucose production were measured with a colorimetric glucose assay kit. ThemRNA expression of PEPCK and G6Pase was quantified by real-time PCR. C: Representative protein levels of IR and FBXO2 in MPHsoverexpressing 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.

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in MPHs (Fig. 7A and Supplementary Fig. 6A–D), suggestingthat inflammation might be responsible for the upregulationof FBXO2 in obese mice. To confirm this point, we deletedKupffer cells in HFD-fed mice by administering GdCl3 (19).Consistent with previous reports that Kupffer cells are theprimary source for hepatic inflammation in obesity (19–21),GdCl3 treatment significantly reduced the expression of pro-inflammatory markers including TNF-a, IL-1b, and F4/80 inliver tissues (Supplementary Fig. 6E). Under this condition,there was a marked decrease in FBXO2 expression in thelivers of obese mice compared with controls (Fig. 7B).

Growing evidence has noted the roles of inflammation-mediated c-Jun N-terminal kinase (JNK) 1 and IKKb/NF-kBsignaling pathways on the regulation of liver metabolic ho-meostasis (20,22–24). Hence, it is interesting to determinewhether JNK1 and/or IKKb/NF-kB activation may underliethe upregulation of FBXO2. As shown in Supplementary Fig.7A, FBXO2 mRNA expression showed similar changes afterTNF-a treatment in JNK1 knockout MPHs compared withJNK1 WT MPHs, suggesting that JNK1 might not be essen-tial for the regulation of FBXO2 expression. In agreementwith this, the induction of FBXO2 was largely blocked by

BAY 11–7082 (an NF-kB inhibitor), but not SP600125 (aJNK inhibitor) or U0126 (an extracellular signal–regulatedkinase inhibitor) (Supplementary Fig. 7B), suggesting thatthe canonical IKKb/NF-kB pathway mediates the effects ofproinflammatory cytokines to induce FBXO2 expression.

Next, we speculated that FBXO2 is a molecular target ofIKKb/NF-kB. To confirm this, we examined the promoterregion of FBXO2 and found that a canonical NF-kB DNA-binding motif (59-GGGRNNYYCC-39) exists in the proximalpromoter region of the FBXO2 gene (Fig. 7C). We thencreated luciferase plasmids controlled by the FBXO2promoter and found that IKKb increased the transcrip-tional activities of these promoters when transfected intoHEK293T cells (Fig. 7D). On the other hand, mutagenesisof the NF-kB DNA-binding motif abrogated the effect ofIKKb/NF-kB in activating the transcriptional activities ofthese promoters (Fig. 7D). Similarly, inhibition of NF-kBactivation by BAY 11–7082 abolished the TNF-a–inducedactivity of the FBXO2 promoter (Fig. 7E), further suggest-ing that hepatic inflammation regulates FBXO2 throughNF-kB signaling. The association of p65 with the FBXO2promoter was also confirmed by ChIP assays (Fig. 7F).

Figure 5—Overexpression of FBXO2 impairs the hepatic actions of insulin and induces hyperglycemia in C57BL/6 mice. A: RepresentativeWestern blots showing levels of FBXO2 protein in the liver of C57BL/6 mice at day 14 after infection with adenoviruses encoding FBXO2or green fluorescent protein (GFP) control. B–D: Blood glucose (B) and insulin (C ) concentrations and GTTs and ITTs (D) in C57BL/6mice. 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 acuteinsulin injection in C57BL/6 mice. Mice were fasted overnight and injected intraperitoneally with insulin (0.75 U insulin/kg body weight) orsaline. After injection (10 min), liver tissues were harvested for homogenization. F: Relative mRNA levels of PEPCK and G6Pase from twogroups of mice (n = 8). **P < 0.01; ***P < 0.001. IB, immunoblotting.

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Considering these data together, we speculate that chronichepatic inflammation–mediated IKKb/NF-kB activationmay be an important mechanism leading to upregulationof FBXO2 in obesity.

DISCUSSION

Previous studies have created mice, via the Cre-loxP system,with tissue-specific disruption of the IR gene. Intriguingly,hyperglycemia and insulin resistance were only exhibited inliver-specific IR knockout mice and not in skeletal muscle–or fat-specific IR knockout mice (25–27), suggesting thathepatic IR has a critical role in regulating glucose homeo-stasis and insulin sensitivity. Although downstream signal-ing pathways of insulin are well established, moleculardeterminants that directly regulate IR expression remainpoorly elucidated. In this study we provide in vitro andin vivo evidence showing a critical role of FBXO2 as a post-transcriptional regulator of hepatic insulin signaling. First, aprotein purification approach combined with the high-performance liquid chromatography/tandem MS assay wasused to identify IR as a novel interacting protein of FBXO2,which was further confirmed by coimmunoprecipitationassays. FBXO2 interacts with the IR to enhance itsubiquitination-mediated protein degradation. Second, thephysiological role of FBXO2 is further revealed by bothgain-of-function and loss-of-function studies of mice. Over-expression of FBXO2 in the liver led to hyperglycemia,hyperinsulinemia, glucose intolerance, and insulin resistancein healthy mice, whereas selective knockdown of FBXO2 inobese mice improved these symptoms. Third, FBXO2 was

upregulated in obese livers, suggesting that inhibiting theexpression or activity of FBXO2 might represent a potentialtherapeutic target for enhancing insulin sensitivity.

Several studies since the 1970s have reported theabnormal number and function of IRs in various tissuesof insulin-resistant mice, including liver, adipose tissue,skeletal muscle, leukocytes, and endothelial cells, whereasits mRNA levels were not found to be decreased (28–31).These results suggest that the small number of receptorscould be due to posttranscriptional levels. Indeed, it hasbeen shown that IR protein expression could be targetedand inhibited by several microRNAs in adipocytes, heart,and liver (32–34). Song et al. (35) demonstrated that IR isubiquitinated by Mitsugumin 53 (MG53) in skeletal musclebecause IR ubiquitination and insulin-elicited downstreamsignaling are inversely changed in MG53 transgenic miceand MG53 knockout mice. A recent study identified nu-clear ubiquitous casein and cyclin-dependent kinase sub-strate as regulators of IR expression, thereby regulatingenergy homeostasis and glucose metabolism (36). There-fore, along with these studies, molecular interventions thatselectively increase IR expression might provide an attrac-tive avenue to treat T2DM. Although both we and anothergroup (35) found that proteasome inhibitor administrationcould efficiently prevent the degradation of IR by differentE3 ligases, how IR gets into the proteasome for degra-dation remains unclear. Moreover, our bioinformaticsanalyses showed that the glycoproteins that interactedexclusively with WT FBXO2 were highly enriched in themembrane, ER, and lysosome, suggesting other membrane

Figure 6—Knockdown of FBXO2 alleviates the diabetic phenotype in db/db obese mice. A: Quantitative real-time PCR and Western blotanalysis 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 FBXO2shRNA or LacZ shRNA (n = 8 or 9). B–D: Blood glucose (B) and insulin (C ) concentrations and GTTs and ITTs (D) in db/db mice. Data wereobtained 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 inresponse to acute insulin injection in db/dbmice. Mice were fasted overnight and injected intraperitoneally with insulin (0.75 U insulin/kg bodyweight) or saline for 10 min. F: Relative mRNA levels of PEPCK and G6Pase from two groups of db/dbmice (n = 8 or 9). **P< 0.01; ***P< 0.001.IB, immunoblotting.

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glycoproteins might also be ubiquitinated by FBXO2. Mem-brane proteins are subject to a complex series of sorting,trafficking, quality control, and quality maintenance sys-tems, which are largely controlled by ubiquitination (37).Retrotranslocation of misfolded membrane proteins fromthe ER into the cytoplasm and processive cleavage by the26S proteasome also participate in ubiquitination-mediateddegradation (38). Interestingly, it has been reported thatFBXO2 ubiquitinates N-glycosylated proteins that aretranslocated from the ER to the cytosol and functionsin an ER-associated degradation pathway (14). Therefore,the degradation of IR might take place in the ER via retro-translocation, which needs to be determined in future studies.

Our data also indicate that aberrant expression of FBXO2is attributed, at least in part, to the activation of IKKb/NF-kB by proinflammatory factors. Numerous studies havedemonstrated that low-grade and chronic inflammationplays a positive role in the glucose intolerance and insulinresistance seen in obesity (39). While several potential mech-anisms have been proposed (39), our results may provide a

novel insight whereby inflammation inhibits the hepaticactions of insulin. In addition, whether FBXO2 expressioncould be regulated by other factors such as ER stress andautophagy remains to be determined.

To our knowledge, we have for the first time identifiedFBXO2 as a functional E3 ligase for IR in the liver. Severalrecent reports showed that FBXO2 plays an important rolein the brain by controlling the abundance of the N-methyl-D-aspartate receptor and amyloid precursor protein (40,41).However, its role in other biological events remainslargely unexplored. Therefore, future studies directedat understanding its tissue-specific downstream targetsare needed.

Acknowledgments. The authors are grateful to Xiaoying Li fromZhongshan Hospital, Fudan University, Shanghai, for helpful discussion ofthe manuscript.Funding. This study was supported by grants from the National NaturalScience Foundation of China (grant nos. 81402478, 31401185, and 81570769), the

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. Micewere 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 mouseFBXO2 gene contains a potential binding site for NF-kB. D and E: Luciferase reporter assays. HEK293T cells were transfected withluciferase 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 weretreated 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.

diabetes.diabetesjournals.org Liu and Associates 697

Shanghai Rising-Star Program (grant no. 16QA1402900), and the ResearchFoundation of Hubei Polytechnic University for Talented Scholars (grant no. 9666).Duality of Interest. No potential conflicts of interest relevant to this articlewere reported.Author Contributions. B.L., H.L., and L.G. performed animal and cellularexperiments and analyzed the data. D.L. and X.X. provided technical advice onthe cellular studies. Z.W. and Y.L. conceived the research ideas, supervised theproject, and wrote the manuscript. Y.L. is the guarantor of this work and, assuch, had full access to all the data in the study and takes responsibility for theintegrity of the data and the accuracy of the data analysis.

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698 Ubiquitin-Mediated FBXO2 Disrupts Homeostasis Diabetes Volume 66, March 2017