Page 1 of 48 Diabetes 1 Aberrant expression of FBXO2 disrupts glucose homeostasis through ubiquitin-mediated degradation of insulin receptor in obese mice Bin Liu1 *, Han Lu2 *, Duanzhuo Li1, Xuelian Xiong3, Lu Gao4, 5, Zhixiang Wu6, and Yan Lu1, 3 1 Hubei Key Laboratory for Kidney Disease Pathogenesis and Intervention, Huangshi Cental Hospital of Edong Healthcare Group, Hubei Polytechnic University School of Medicine, Huangshi, Hubei 435003, PR China. 2 Department of Anesthesiology, Ruijin Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200025, PR China. 3 Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, Shanghai 200032, PR China. 4 College of Life Sciences, Northeast Agricultural University, No.59 Mucai street, Harbin 150030, Heilongjiang, PR China. 5 Department of Pathology, University of Maryland School of Medicine, 655W. Baltimore Street, Baltimore, MD, 21202-1192,USA. 6 Department of Pediatric Surgery, Xinhua Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), Shanghai 200092, PR China. * Co-first author 1 Diabetes Publish Ahead of Print, published online December 8, 2016 Diabetes Page 2 of 48 2 Corresponding authors Dr. Lu Gao, College of Life Sciences, Northeast Agricultural University, No.59 Mucai street, Harbin 150030, Heilongjiang, PR China. Tel: +86-045155191257 Fax: +86-045155191257 E-mail: [email protected] Dr. Zhixiang Wu, Department of Pediatric Surgery, Xinhua Hospital, Shanghai Jiao-Tong University School of Medicine (SJTU-SM), 1665 Kongjiang Road, Shanghai 200092, PR China. Tel: +86-21-25078413, Fax: +86-21-65791316, E-mail: [email protected] Dr. Yan Lu, Department of Endocrinology and Metabolism, Zhongshan Hospital, Fudan University, 180 Fenglin Road, Shanghai 200032, PR China. Tel: +86-21-64041990 Fax: +86-21-64041990 E-mail: [email protected] 2 Page 3 of 48 Diabetes 3 Abstract Insulin resistance is a critical factor in the development of metabolic disorders, including type 2 diabetes (T2DM). However, its molecular mechanisms remain incompletely understood. In the present study, we found that F-box only protein 2 (FBXO2), a substrate recognition component of SKP1-Cullin1-F-box protein (SCF) E3 ubiquitin ligase complex, were up-regulated in livers of obese mice. Furthermore, using a protein purification approach combined with high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS), we carried out a system-wide screening of FBXO2 substrates, in which insulin receptor (IR) was identified as a substrate for FBXO2. SCFFBXO2 acts as an E3 ligase targeting the IR for ubiquitin-dependent degradation to regulate insulin signaling integrity. As a result, adenovirus-mediated overexpression of FBXO2 in healthy mice led to hyperglycemia, glucose intolerance and insulin resistance, while ablation of FBXO2 alleviated diabetic phenotypes in obese mice. Therefore, our results identify SCFFBXO2 as an E3 ligase for IR in the liver, which might provide a novel therapeutic target for treating T2DM and related metabolic disorders. Key Words: Type 2 diabetes, Insulin resistance, Insulin signaling, 3 Diabetes Page 4 of 48 4 Ubiquitination, Protein degradation, Type 2 diabetes mellitus (T2DM), characterized by high blood glucose levels, has become a pandemic problem worldwide. Usually, hyperglycemia is caused by deficiency of insulin secretion and/or reduced insulin sensitivity. In peripheral tissues, including liver, skeletal muscle and adipose tissue, insulin binds to its receptor (IR), which then phosphorylates and recruits insulin receptor substrates (IRS) to further activate downstream signaling pathways (1). In the liver, the major node of insulin signaling is activation of phosphoinositide-3-kinase (PI3K)/AKT, which in turn inhibits the expression of phosphoenolpyruvate carboxykinase (PEPCK) and glucose 6-phosphatase (G6Pase), two key gluconeogenic enzymes (2). As a result, hepatic insulin resistance is characterized by excessive hepatic glucose production, contributing to fasting hyperglycemia in T2DM (3). Therefore, identification of novel molecules involved in regulating the hepatic insulin signaling pathway will advance our understanding of the pathogenesis that leads to T2DM. Polyubiquitination is the formation of an ubiquitin chain 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 4 Page 5 of 48 Diabetes 5 reactions: activation, conjugation, and ligation, performed by ubiquitin-activating enzymes (E1s), ubiquitin-conjugating enzymes (E2s), and ubiquitin ligases (E3s), respectively (5). The largest subfamily of E3s in mammalian is the Skp1-Cul1-F box protein ubiquitin ligases (SCFs), which consist of Skp1, Cul1, Rbx1, and one of F box proteins (FBPs) (6). Recent studies have shown that FBPs play a crucial role in many biological events, such as inflammation, cell-cycle progression and tumorigenesis, through ubiquitin-mediated degradation of cellular regulatory proteins (7; 8). Besides, their dysregulation has been implicated in several pathologies (6-8), suggesting that insights into SCF-mediated biology may provide potential strategies to treat human diseases. However, until now, whether FBPs play a role in the metabolic diseases, especially insulin resistance and T2DM, remains poorly understood. Research design and Methods Animal experiments Male C57BL/6 and db/db mice aged 8-10 weeks were purchased from the Shanghai Laboratory Animal Company (SLAC) and Nanjing Biomedical Research Institute of Nanjing University, respectively. JNK1 knockout 5 Diabetes Page 6 of 48 6 mice were obtained from Jackson Laboratories and backcrossed to C57BL/6 background for 6 generations. All mice were housed at 21°C ± 1°C with humidity of 55% ± 10% and a 12-hour light/12-hour dark cycle. HFD-induced obese mice were maintained with free access to high-fat chow (D12492; Research Diets) containing 60% kcal from fat, 20% kcal from carbohydrate, and 20% kcal from protein. For the depletion of Kupffer cells, C57BL/6 mice were fed with a high-fat-diet for 12 weeks and then injected with gadolinium chloride (GdCl3, 10 mg/kg, twice each week) or sodium chloride (NaCl) by tail vein for another 2 weeks. All study protocols comply with guidelines and institutional policies prepared by the Animal Care Committee of Shanghai Jiao Tong University School of Medicine. Immuoprecipitation (IP) and in-solution digestion The standard IP purification procedure has been previously described (9). In brief, HEK293T cells stably expressing Flag-tagged wild-type or mutant FBXO2 were lysed in 5 mL lysis buffer (50 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.5% Nonidet P40, and 100mM PMSF) for 20 min with gentle rocking at 4 °C. Lysates were cleared and subjected to IP with 50 μL of anti-FLAG M2 beads overnight at 4 °C. Beads containing immune 6 Page 7 of 48 Diabetes 7 complexes were washed with 1 mL ice cold lysis buffer. Proteins were eluted with 100 μL 3 × Flag-peptide (Sigma-Aldrich, St. Louis, MO, USA) in TBS for 30 min and precipitated with cold acetone. The precipitated proteins were in-solution digested with trypsin, and the tryptic peptides were vacuum centrifuged to dryness for further analysis. HPLC/MS/MS analysis Nanoflow LC-MS/MS was performed by coupling an Easy nLC 1000 (Thermo Fisher Scientific, Waltham, MA) to an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Waltham, MA). Tryptic peptides were dissolved in 20 µL of 0.1% formic acid, and 10 µL were injected for each analysis. Peptides were delivered to a trap column (2 cm length with 100 µm inner diameter, packed with 5 µm C18 resin) at a flow rate of 5 µL/min in 100% buffer A (0.1% FA in HPLC grade water). After 10 min of loading and washing, the peptides were transferred to an analytical column (17 cm× 79 μm, 3-μm particle size, Dikma, China) coupled to Easy nLC 1000 (Thermo Fisher Scientific, Waltham, MA). The separated peptides were ionized using NSI source, then analyzed in an Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with a top speed 3s data-dependent mode. For MS/MS scan, ions with 7 Diabetes Page 8 of 48 8 intensity above 5,000 and charge state 2-6 in each full MS spectrum were sequentially fragmented by Higher Collision Dissociation with normalized collision energy of 32%. The dynamic exclusion duration was set to be 60 s, and the precursor ions were isolated by quadrupole with isolation window 1 Da. The fragment ions were analyzed in ion trap with AGC 7,000 at rapid scan mode. The raw spectra data was processed by protein discover and MS/MS spectra data was searched against the Uniprot human database (88,817 sequences) by Mascot (v.2.4, Matrix Science, London, UK) Bioinformatics analysis The molecular function, cellular component analysis of the glycoproteins was performed using Database for Annotation, Visualization and Integrated Discovery Bioinformatics Database (DAVID 6.7) (10; 11). Glucose and insulin tolerance tests Glucose tolerance tests were performed by intraperitoneal injection of D-glucose (Sigma-Aldrich, USA) at a dose of 2.0 mg/g body weight after a 16-hour fast. For insulin tolerance tests, mice were injected with regular human insulin (Eli Lily, Indianapolis, Indiana, USA) at a dose of 0.75 U/kg 8 Page 9 of 48 Diabetes 9 body weight after a 6-hour fast. Blood glucose was measured by a portable blood glucose meter (Lifescan, Johnson & Johnson, New Brunswick, New Jersey, USA). Western blots Hepatic tissues or cells were lysed in radioimmunoprecipitation buffer containing 50 mM Tris-HCl, 150 mM NaCl, 5 mM MgCl2, 2 mM EDTA, 1 mM NaF, 1% NP-40, and 0.1% sodium dodecyl sulfate. Western blots were performed using antibodies against FBXO2 (ab133717; Abcam), IRβ (ab131238; Abcam), AKT (13038, 4821; Cell Signaling) and GAPDH (5174; Cell Signaling). Analysis of tyrosine phosphorylation of IRS1 was performed by immunoprecipitation of IRS1 with anti-IRS1 from total lysate, followed by western blot with anti-pTyr antibody (PY100). Luciferase reporter and Chromatin immunoprecipitation assays All the transient transfections were conducted using Lipofectamine 2000 (Invitrogen, Shanghai, China).