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

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Aberrant Expression of FBXO2 Disrupts Glucose Homeostasis Through Ubiquitin-Mediated Degradation of Insulin Receptor in Obese Mice 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 Ubiquitin-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 protein phosphorylates and recruits IR substrates (IRSs) to further 2 (FBXO2), a substrate recognition component of the activate downstream signaling pathways (1). In the liver, OBESITY STUDIES Skp1-Cul1-F-box protein (SCF) E3 ubiquitin ligase 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 proteins (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).
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