O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes

Abstract

Abbreviations: GLUT, glucose transporter; HBP, hexosamine biosynthetic pathway; IRS, insulin receptor substrate; PDK-1, phosphatidylinositol dependent protein kinase-1; PKC-λ/ζ, protein kinase C-λ/ζ; PI3K, phospatidylinositol 3-kinase; PM, plasma membrane IntroductionThe pathophysiology of non-insulin-dependent diabetes mellitus (NIDDM) results from impaired peripheral tissue sensitivity to insulin and reduction of insulin secretion. Glucose uptake in mammalian cells is mediated by a family of intrinsic membrane proteins known as facilitative glucose transporters, GLUTs (Mueckler, 1994). GLUT4, the insulin-responsive glucose transporter, is selectively expressed in adipocytes and muscle (Bryant et al., 2002). GLUT4 in these cells constantly recycles between the plasma membrane (PM) and intracellular storage pools (Cushm an and W ardzala, 1980). About 90 percent of GLUT4 is sequestered intracellularly in the absence of insulin or other stimuli such as exercise (Cushman and Wardzala, 1980). Insulin stimulates glucose uptake in these cells primarily by inducing net translocation of GLUT4 from the intracellular storage sites to the PM (Lee et al., 1999, Lee et al., 2000b. The translocation of GLUT4 by insulin results from a cascade of signal transduction, which is composed of a series of molecules (Pessin et al., 1999). Insulin-stimulated GLUT4 translocation and glucose uptake are mediated largely through the activation/phosphorylation of IRS-1 and its downstream effectors, such as PI3K, PDK-1, PKC-λ/ζ, and Akt2 (Saltiel and Pessin, 2002;Kanzaki and Pessin, 2003). An impaired GLUT4 translocation and/or insulin signaling pathway would result in insulin resistance and hyperglycemia, the primary characteristics and hallmarks of NIDDM (Watson and Pessin, 2001). O-GlcNAc m odification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat prim ary adipocytes O-GlcNAc modification and insulin resistance 221It has been known that O-GlcNAc modification of protein plays an important role in transcription, translation, nuclear transport and cytoskeletal assembly (Comer and Hart, 2000;Wells et al., 2001). Recently, O-GlcNAc modification as well as phosphorylation/ dephosphorylation has been focused on as a main regulation mechanism of cellular signal transduction pathways. Post-translational O-GlcNAc modification on proteins is defined as the O-linked attachment of single GlcNAc moiety to specific hydroxyl groups of Ser or Thr residue (Buse et al., 2002). Since Oglycosylation of cellular proteins via Ser/Thr takes place dynamically in various ways and the modification site is usually located near the O-phosphorylation site or sometimes at the same site, O-GlcNAc modification plays as a dynamic negative regulator against protein phosphorylation (Snow and Hart, 1998, Zachara and. Therefore, O-glycosylation and O-phosphorylation are known to participate in the modification of protein interaction and regulation of signal transduction by changing the protein reaction site through the mutua...

Caenorhabditis elegans ortholog of a diabetes susceptibility locus: oga-1 ( O -GlcNAcase) knockout impacts O -GlcNAc cycling, metabolism, and dauer

“…The C. elegans oga-1(ok1207) mutant exhibits increased levels of O-GlcNAc on nuclear pores and other cellular targets and alterations in macronutrient storage and intracellular signaling. In mammals, reduction of O-GlcNAcase activity produces insulin resistance in tissue culture and isolated tissues (39,40,61). These findings, coupled with our previous findings that OGT overexpression produces insulin resistance in mice (17), demonstrate a role for O-GlcNAc cycling in metabolism and insulin signaling.…”

“…The oga-1(ok1207) mutation enhanced dauer formation in the sensitized daf-2 mutant background. This resistance to insulin-like signaling in the OGA-1 knockout is highly reminiscent of studies in mammalian cells where enhanced O-GlcNAc levels induced by OGT overexpression (17) or treatment with OGA inhibitors produce insulin resistance (17,39,40,61,80). In contrast, the ogt-1(ok430) mutation displayed hypersensitivity to insulin-like signaling in the dauer assay, suggesting that OGT-1 normally acts to blunt insulin-like signaling (26).…”

“…One of the striking features of type 2 diabetes mellitus is insulin resistance. Resistance to insulin is associated with increased flux through the hexosamine biosynthetic pathway (28)(29)(30)(31)(32)(33) as well as with inhibition of OGA (34)(35)(36)(37)(38)(39)(40)(41)(42). The link between hexosamine signaling and human diabetes mellitus was more directly shown by the finding that a single nucleotide polymorphism in the MGEA5 gene encoding OGA is associated with diabetes mellitus and age of onset in the Mexican-American population (43).…”

O-GlcNAc Regulates FoxO Activation in Response to Glucose

“…5), indicating that additional, intranuclear mechanisms of transactivation exist. It is possible that this mechanism involves GlcNAcylation of upstream regulators of FoxO because AKT and insulin receptor substrate 1 are also GlcNAcylated (47). This mechanism is unlikely, however, because the mutation of a very specific hydroxyamino acid (Thr 317 but not Ser 318 ), which we have shown to be GlcNAcylated, reduces FoxO1 stimulation by hyperglycemia.…”

Regulation of Insulin Receptor Substrate 1 (IRS-1)/AKT Kinase-mediated Insulin Signaling by O-Linked β-N-Acetylglucosamine in 3T3-L1 Adipocytes

“…Several groups have shown that O-GlcNAc directly inhibits insulin signaling in several cell lines (16 -18). In addition, there are a number of other publications that suggest that the role of O-GlcNAc in Type II diabetes may be cell and protein cell signaling specific to adipocytes (16,25,49), muscles (18), and human coronary artery endothelial cells (17) because others have found that O-GlcNAc did not have any affect on AKT phosphorylation in neuroblastoma cells (50), neural precursor cells (51), and skeletal muscle cells (52). Interestingly, OGT itself, the enzyme responsible for mediating the addition of O-GlcNAc, is known to be involved in insulin signaling (13,24,25), activated by insulin signaling (13), and involved in insulin resistance (24,25).…”