title: Terminal Galactosylation and Sialylation Switching on Membrane Glycoproteins upon TNF-Alpha-Induced Insulin Resistance in Adipocytes.
abstract: Insulin resistance (IR) is a complex pathophysiological state that arises from both environmental and genetic perturbations and leads to a variety of diseases, including type-2 diabetes (T2D). Obesity is associated with enhanced adipose tissue inflammation, which may play a role in disease progression. Inflammation modulates protein glycosylation in a variety of cell types, and this has been associated with biological dysregulation. Here, we have examined the effects of an inflammatory insult on protein glycosylation in adipocytes. We performed quantitative N-glycome profiling of membrane proteins derived from mouse 3T3-L1 adipocytes that had been incubated with or without the proinflammatory cytokine TNF-alpha to induce IR. We identified the regulation of specific terminal N-glycan epitopes, including an increase in terminal di-galactose- and a decrease in biantennary alpha-2,3-sialoglycans. The altered N-glycosylation of TNF-alpha-treated adipocytes correlated with the regulation of specific glycosyltransferases, including the up-regulation of B4GalT5 and Ggta1 galactosyltransferases and down-regulation of ST3Gal6 sialyltransferase. Knockdown of B4GalT5 down-regulated the terminal di-galactose N-glycans, confirming the involvement of this enzyme in the TNF-alpha-regulated N-glycome. SILAC-based quantitative glycoproteomics of enriched N-glycopeptides with and without deglycosylation were used to identify the protein and glycosylation sites modified with these regulated N-glycans. The combined proteome and glycoproteome workflow provided a relative quantification of changes in protein abundance versus N-glycosylation occupancy versus site-specific N-glycans on a proteome-wide level. This revealed the modulation of N-glycosylation on specific proteins in IR, including those previously associated with insulin-stimulated GLUT4 trafficking to the plasma membrane.
authors.affiliation: From the ‡Charles Perkins Centre, School of Molecular Bioscience and.
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Parker
authors.affiliation: ¶Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Morten
authors.initials: M
authors.lastname: Thaysen-Andersen
authors.affiliation: From the ‡Charles Perkins Centre, School of Molecular Bioscience and.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Fazakerley
authors.affiliation: From the ‡Charles Perkins Centre, School of Molecular Bioscience and.
authors.forename: Mira
authors.initials: M
authors.lastname: Holliday
authors.affiliation: ¶Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.affiliation: From the ‡Charles Perkins Centre, School of Molecular Bioscience and §School of MedicineUniversity of Sydney, Sydney, Australia; david.james@sydney.edu.au.
authors.forename: David E
authors.initials: DE
authors.lastname: James
mesh_terms: D041721:3T3-L1 Cells; D017667:Adipocytes / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism*; D000818:Animals; D005690:Galactose / Q000378:metabolism; D054794:Glycomics / Q000379:methods; D006020:Glycopeptides / Q000378:metabolism; D006031:Glycosylation; D007333:Insulin Resistance; D013058:Mass Spectrometry; D008562:Membrane Glycoproteins / Q000378:metabolism*; D051379:Mice; D019158:N-Acetylneuraminic Acid / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D040901:Proteomics / Q000379:methods; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
chemical_list: D006020:Glycopeptides; D008562:Membrane Glycoproteins; D011134:Polysaccharides; D020543:Proteome; D014409:Tumor Necrosis Factor-alpha; D019158:N-Acetylneuraminic Acid; D005690:Galactose