O-linked (3-N-acetylglucosamine(O- GlcNAc) is a dynamic and inducible post-translational modification that is covalently attached to serine and threonine residues on nuclear and cytoplasmic proteins. We and others have demonstrated that elevation in the levels of O-GlcNAc transferred to intracellular proteins induces insulin resistance, the hallmark of Type II diabetes.
The specific aims of this proposal will test the hypothesis that insulin action is inhibited by elevated O-GlcNAc modification of specific proteins in the insulin signal transduction cascade. In three different model systems, we have determined that the defect lies in the metabolic branch of insulin signaling, downstream of the receptor and at or upstream of the protein kinase AKT. We have developed several essential tools for the study of the O-GlcNAc post- translational modification in insulin signal transduction. These include an O-GlcNAc specific monoclonal antibody, cell lines for the inducible expression of enzymes that add and remove O-GlcNAc, C. elegans strains that harbor deletions in the O-GlcNAc cycling enzymes, and a tandem mass spectrometry-based approach for site-mapping and quantification of post-translational modifications.
In Specific Aim 1, we will expand on our preliminary findings to demonstrate that inappropriate O-GlcNAc modification perturbs insulin signaling as measured by changes in apoptosis, glucose-uptake, and/or lifespan using mammalian cell lines and C. elegans as model systems. Furthermore, functional changes (activity, localization, associations, and phosphorylation) in proteins of the metabolic branch of the insulin pathway upon perturbations in global O-GlcNAc levels will be elucidated.
In Specific Aim 2, we will identify and site- map O-GlcNAc modified proteins associated with the insulin signaling pathway using our suite of tandem mass spectrometry-based approaches.
In Specific Aim 3, we will assign functional consequences to O- GlcNAc modification of specific signal transduction proteins in the metabolic branch of the insulin cascade. By reintroducing tagged glycosylation-competent wild type and glycosylation-incompetent mutant proteins back into mammalian cell lines and into C. elegans strains that are null for the protein of interest, we will determine how differential O-GlcNAc modification of specific sites on particular proteins affects insulin signaling. Completion of these aims will elucidate both global and molecular consequences of protein O- GlcNAc modification with regard to regulating insulin action in mammalian cell lines and in C. elegans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK075069-02
Application #
7336329
Study Section
Intercellular Interactions (ICI)
Program Officer
Haft, Carol R
Project Start
2007-01-01
Project End
2011-12-31
Budget Start
2008-01-01
Budget End
2008-12-31
Support Year
2
Fiscal Year
2008
Total Cost
$267,418
Indirect Cost
Name
University of Georgia
Department
Type
Organized Research Units
DUNS #
004315578
City
Athens
State
GA
Country
United States
Zip Code
30602
Zhao, Peng; Stalnaker, Stephanie H; Wells, Lance (2013) Approaches for site mapping and quantification of O-linked glycopeptides. Methods Mol Biol 951:229-44
Wells, Lance; Slawson, Chad; Hart, Gerald W (2011) The E2F-1 associated retinoblastoma-susceptibility gene product is modified by O-GlcNAc. Amino Acids 40:877-83
Zhao, Peng; Viner, Rosa; Teo, Chin Fen et al. (2011) Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment. J Proteome Res 10:4088-104
Rahman, Mohammad M; Stuchlick, Olga; El-Karim, Enas G et al. (2010) Intracellular protein glycosylation modulates insulin mediated lifespan in C.elegans. Aging (Albany NY) 2:678-90
Brimble, Sandii; Wollaston-Hayden, Edith E; Teo, Chin Fen et al. (2010) The Role of the O-GlcNAc Modification in Regulating Eukaryotic Gene Expression. Curr Signal Transduct Ther 5:12-24
Teo, Chin Fen; Ingale, Sampat; Wolfert, Margreet A et al. (2010) Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. Nat Chem Biol 6:338-43
Teo, Chin Fen; Wollaston-Hayden, Edith E; Wells, Lance (2010) Hexosamine flux, the O-GlcNAc modification, and the development of insulin resistance in adipocytes. Mol Cell Endocrinol 318:44-53
Fang, Meng; Lim, Jae-Min; Wells, Lance (2010) Quantitative Glycomics of Cultured Cells Using Isotopic Detection of Aminosugars with Glutamine (IDAWG). Curr Protoc Chem Biol 2:55-69
Orlando, Ron; Lim, Jae-Min; Atwood 3rd, James A et al. (2009) IDAWG: Metabolic incorporation of stable isotope labels for quantitative glycomics of cultured cells. J Proteome Res 8:3816-23
Webster, Danielle M; Teo, Chin Fen; Sun, Yuhua et al. (2009) O-GlcNAc modifications regulate cell survival and epiboly during zebrafish development. BMC Dev Biol 9:28