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O-Glcnac Modification on IRS-1 and Akt2 by Pugnac Inhibits Their Phosphorylation and Induces Insulin Resistance in Rat Primary Adipocytes

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O-Glcnac Modification on IRS-1 and Akt2 by Pugnac Inhibits Their Phosphorylation and Induces Insulin Resistance in Rat Primary Adipocytes EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 37, No. 3, 220-229, June 2005 O-GlcNAc modification on IRS-1 and Akt2 by PUGNAc inhibits their phosphorylation and induces insulin resistance in rat primary adipocytes Seung Yoon Park1, Jiwon Ryu2 and ing that PUGNAc developed impaired glucose Wan Lee1,3 utilization and insulin resistance in adipocytes. Interestingly, the O-GlcNAc modification of IRS-1 1From the Department of Biochemistry and Akt2 was increased by PUGNAc, accom- College of Medicine, Dongguk University panied by a partial reduction of insulin-stimulated Gyeongju 780-714, Korea phosphorylations of IRS-1 and Akt2. The PUGNAc 2Biophysics Laboratory treatment has no effect on the expression level Veteran Affairs Medical Center of GLUT4, whereas O-GlcNAc modification of Department of Physiology and Biophysics GLUT4 was increased. These results suggest that School of Medicine, State University of New York at Buffalo the increase of O-GlcNAc modification on insulin Buffalo, NY 14215, USA signal pathway intermediates, such as IRS-1 and 3Corresponding author: Tel, 82-54-770-2409; Akt2, reduces the insulin-stimulated phosphoryla- Fax, 82-54-770-2447; E-mail, [email protected] tion of IRS-1 and Akt2, subsequently leading to insulin resistance in rat primary adipocytes. Accepted 7 June 2005 Keywords: Akt2; GLUT4; insulin resistance; IRS-1; Abbreviations: GLUT, glucose transporter; HBP, hexosamine bio- O-GlcNAc; PUGNAc synthetic pathway; IRS, insulin receptor substrate; PDK-1, phospha- tidylinositol dependent protein kinase-1; PKC-λ/ζ, protein kinase C-λ/ζ; PI3K, phospatidylinositol 3-kinase; PM, plasma membrane Introduction The pathophysiology of non-insulin-dependent diabe- tes mellitus (NIDDM) results from impaired peripheral Abstract tissue sensitivity to insulin and reduction of insulin secretion. Glucose uptake in mammalian cells is med- It has been known that O-linked β-N-acetylgluco- iated by a family of intrinsic membrane proteins samine (O-GlcNAc) modification of proteins plays known as facilitative glucose transporters, GLUTs an important role in transcription, translation, (Mueckler, 1994). GLUT4, the insulin-responsive glu- nuclear transport and signal transduction. The cose transporter, is selectively expressed in adipo- increased flux of glucose through the hexosa- cytes and muscle (Bryant et al., 2002). GLUT4 in mine biosynthetic pathway (HBP) and increased these cells constantly recycles between the plasma O-GlcNAc modification of protein have been sug- membrane (PM) and intracellular storage pools (Cush- gested as one of the causes in the development man and Wardzala, 1980). About 90 percent of GLUT4 is sequestered intracellularly in the absence of insulin resistance. However, it is not clear at of insulin or other stimuli such as exercise (Cushman the molecular level, how O-GlcNAc protein modi- and Wardzala, 1980). Insulin stimulates glucose up- fication results in substantial impairment of in- take in these cells primarily by inducing net translo- sulin signaling. To clarify the association of O - cation of GLUT4 from the intracellular storage sites GlcNAc protein modification and insulin resist- to the PM (Lee et al., 1999, Lee et al., 2000b). The ance in rat primary adipocytes, we treated the translocation of GLUT4 by insulin results from a cas- cade of signal transduction, which is composed of a adipocytes with O-(2-acetamido-2deoxy-D-gluco- series of molecules (Pessin et al., 1999). Insulin-sti- pyranosylidene)amino-N -phenylcarbamate mulated GLUT4 translocation and glucose uptake are (PUGNAc), a potent inhibitor of O-GlcNAcase that mediated largely through the activation/phosphor- catalyzes removal of O-GlcNAc from proteins. ylation of IRS-1 and its downstream effectors, such Prolonged treatment of PUGNAc (100 µM for 12 as PI3K, PDK-1, PKC-λ/ζ, and Akt2 (Saltiel and h) increased O-GlcNAc modification on proteins Pessin, 2002; Kanzaki and Pessin, 2003). An impaired in adipocytes. PUGNAc also drastically decreased GLUT4 translocation and/or insulin signaling pathway would result in insulin resistance and hyperglycemia, insulin-stimulated 2-deoxyglucose (2DG) uptake the primary characteristics and hallmarks of NIDDM and GLUT4 translocation in adipocytes, indicat- (Watson and Pessin, 2001). O-GlcNAc modification and insulin resistance 221 It has been known that O-GlcNAc modification of increased cellular O-GlcNAc modification of proteins protein plays an important role in transcription, trans- and also induced insulin resistance in 3T3-L1 adipo- lation, nuclear transport and cytoskeletal assembly cytes (Vosseller et al., 2002) and rat skeletal muscle (Comer and Hart, 2000; Wells et al., 2001). Recently, (Arias et al., 2004). These results indicate that either O-GlcNAc modification as well as phosphorylation/ activation of O-GlcNAc transferase or inhibition of O- dephosphorylation has been focused on as a main GlcNAcase increases O-GlcNAc modification on pro- regulation mechanism of cellular signal transduction teins, and subsequently attenuates insulin signaling in pathways. Post-translational O-GlcNAc modification on peripheral tissues such as muscle and adipocytes. proteins is defined as the O-linked attachment of Recently, Vosseller et al. (2002) demonstrated that single GlcNAc moiety to specific hydroxyl groups of PUGNAc induces insulin resistance by reducing Ser or Thr residue (Buse et al., 2002). Since O- insulin-stimulated phosphorylation of Akt and glycogen glycosylation of cellular proteins via Ser/Thr takes synthase kinase 3β (GSK3β) in 3T3-L1 adipocytes. place dynamically in various ways and the modifica- However, Arias et al. (2004) suggest that PUGNAc- tion site is usually located near the O-phosphorylation induced insulin resistance is not attributable to re- site or sometimes at the same site, O-GlcNAc modi- duced phosphorylation of Akt or GSK3α/β in rat fication plays as a dynamic negative regulator against skeletal muscle. There is also a report that the glu- protein phosphorylation (Snow and Hart, 1998, Za- cosamine-induced insulin resistance is closely asso- chara and Hart, 2002). Therefore, O-glycosylation and ciated with increased O-GlcNAc modification and O-phosphorylation are known to participate in the reduced tyrosine phosphorylation of IRS-1 and -2 in modification of protein interaction and regulation of rat skeletal muscle (Patti et al., 1999). This evidence signal transduction by changing the protein reaction revealed that O-GlcNAc modification on proteins by site through the mutual interaction (Griffith and Sch- PUGNAc causes insulin resistance in muscle and mitz, 1999; Cheng et al., 2000). adipocytes (Haltiwanger et al., 1998; Gao et al., 2000; With increasing knowledge about O-GlcNAc modi- Vosseller et al., 2002; Arias et al., 2004). However, fication as a new regulatory signal, there have been it is not clear how and which O-GlcNAc modification reports that abnormalities in the regulation of O- of proteins causes substantial disturbances in glucose GlcNAc protein modification are one of the causes in uptake and insulin signaling, leading to insulin re- the pathogenesis of NIDDM. Marshall et al. (1991) sistance in rat adipocytes. first implicated HBP in glucose-induced insulin re- In the present study, we report that treatment of sistance by demonstrating a requirement for the en- PUGNAc is directly correlated with the elevation of zyme, glutamine fructose 6-phosphate amidotrans- O-GlcNAc protein modification and insulin resistance ferase (GFAT). The metabolic step catalyzed by in rat primary adipocytes. Prolonged treatment of GFAT provides the necessary substrates for protein PUGNAc increased O-GlcNAc modification on several modification by O-GlcNAc. Subsequently, the infusion proteins and partially inhibited insulin-stimulated 2DG of glucosamine induced insulin resistance in rat by uptake and GLUT4 translocation. The O-GlcNAc mod- increasing uridine diphosphate-N-acetylglucosamine ification of IRS-1 and Akt2 was increased by PUGNAc, (UDP-GlcNAc) levels (Rossetti et al., 1995), and the accompanied by a partial reduction of insulin-sti- transgenic mice over-expressing GFAT also devel- mulated phosphorylations of IRS-1 and Akt2. It is oped insulin resistance with high GFAT activity and suggested that the increase of O-GlcNAc modification UDP-GlcNAc levels (Hebert et al., 1996). O-Glyco- on insulin signal pathway intermediates, such as sylation of several proteins was increased by the IRS-1 and Akt2, and the reduction in insulin-stimul- O-GlcNAc infusion in rats (Yki-Jarvinen et al., 1998), ated phosphorylation of IRS-1 and Akt2 are assoc- and insulin-stimulated GLUT4 translocation to the PM iated with the insulin resistance developed in PUGNAc- was inhibited by high glucosamine treatment in rat treated rat primary adipocytes. skeletal muscle (Baron et al., 1995). In addition to UDP-GlcNAc flux, the overexpression of O-GlcNAc transferase, an enzyme catalyzing the addition of O- Materials and Methods GlcNAc on target proteins, induced insulin resistance and hyperleptinemia in transgenic mice (McClain et Materials al., 2002). This result suggests that GlcNAc trans- ferase participates in a hexosamine-dependent sig- Collagenase type I was obtained from Worthington naling pathway that is linked to insulin resistance and (Lakewood, NJ). The anti-O-GlcNAc antibody desig- leptin production. O-GlcNAcase, an nucleocytoplasmic nated as CTD110.6 was purchased from Covance enzyme catalyzing removal of O-GlcNAc from pro- (Princeton, NJ). Anti-GLUT4 antibody (clone 1F8) was teins, is
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