Regulation of insulin receptor substrate 1 (IRS-1)/AKT kinase-mediated insulin signaling by O-Linked beta-N-acetylglucosamine in 3T3-L1 adipocytes

Abstract

Increased O-linked beta-N-acetylglucosamine (O-GlcNAc) is associated with insulin resistance in muscle and adipocytes. Upon insulin treatment of insulin-responsive adipocytes, O-GlcNAcylation of several proteins is increased. Key insulin signaling proteins, including IRS-1, IRS-2, and PDK1, are substrates for OGT, suggesting potential O-GlcNAc control points within the pathway. To elucidate the roles of O-GlcNAc in dampening insulin signaling (Vosseller, K., Wells, L., Lane, M. D., and Hart, G. W. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 5313-5318), we focused on the pathway upstream of AKT. Increasing O-GlcNAc in 3T3-L1 adipocytes decreases phosphoinositide 3-kinase (PI3K) interactions with both IRS-1 and IRS-2. Elevated O-GlcNAc also reduces phosphorylation of the PI3K p85 binding motifs (YXXM) of IRS-1 and results in a concomitant reduction in tyrosine phosphorylation of Y(608)XXM in IRS-1, one of the two main PI3K p85 binding motifs. Additionally, insulin signaling stimulates the interaction of OGT with PDK1. We conclude that one of the steps at which O-GlcNAc contributes to insulin resistance is by inhibiting phosphorylation at the Y(608)XXM PI3K p85 binding motif in IRS-1 and possibly at PDK1 as well.

FIGURE 1.

IRS-1, IRS-2, and PDK1 of the PI3K/AKT insulin signaling cascade are OGT substrates. Proteins from the insulin signaling cascade, IRS-1, IRS-2, PI3K p85, PDK, AKT, PKCζ, and GSK3β were immunoprecipitated from 3T3-L1 adipocytes stimulated or not with 100 nm insulin and incubated for 4 h while shaking with 0.5 μg of OGT and OGT reaction buffer containing UDP-[³H]GlcNAc. The reaction was stopped with modified Laemmli buffer, subjected to 10% SDS-PAGE, stained with Coomassie Brilliant Blue G-250, treated with EnHance, dried, and subjected to autoradiography at −80 °C for 4 days. Western blots (WB) of each immunoprecipitated protein were conducted.

FIGURE 2.

Elevations of O-GlcNAc in total cell extracts of 3T3-L1 adipocytes inhibit tyrosine phosphorylation of the Tyr(P)⁶⁰⁸ IRS-1 YXXM motif and downstream AKT phosphorylation. Total cell extracts from 3T3-L1 adipocytes pretreated or not with 100 μm PUGNAc, pretreated or not with 200 nm wortmannin, and stimulated or not with 5 nm insulin were Western blotted with Tyr(P)⁶⁰⁸ IRS-1, Thr(P)³⁰⁸ AKT, AKT, and actin antibodies. Data are representative of at least six experiments. Equal protein loading was demonstrated by Western blotting (WB) with anti-AKT and anti-actin antibody. The intensity of Tyr(P)⁶⁰⁸ IRS-1 Western blot analysis was normalized by equal protein loading and normalized to 100% of insulin stimulation using ImageJ. The intensity of Thr(P)³⁰⁸ AKT Western blot intensity was also quantitated with ImageJ.

FIGURE 3.

IRS-1 is modified with O-GlcNAc, and elevations in O-GlcNAc reduce IRS-1 interaction with PI3K p85 and inhibit phosphorylation of the Tyr(P)⁶⁰⁸ IRS-1 YXXM motif. A, IRS-1 was immunoprecipitated (IP) from 3T3-L1 adipocytes pretreated or not with PUGNAc and then stimulated or not with 5 nm insulin and probed for IRS-1, O-GlcNAc RL-2, phospho-YXXM p85, and Tyr(P)⁶¹² IRS-1. The total cell extract was also Western blotted (WB) for all antibodies as a control. B, IRS-1 was immunoprecipitated from 3T3- L1 adipocytes pretreated or not with PUGNAc and labeled with GalT1, UDP-N-azidogalactosamine (GalNAz), and biotin tag and subjected to SDS-PAGE and Western blotting with streptavidin-horseradish peroxidase. C, IRS-1 and PI3K p85 were immunoprecipitated from PUGNAc-treated or -untreated and 5 nm insulin-stimulated or -unstimulated 3T3-L1 adipocytes and Western blotted for PI3K p85 and IRS-1 interaction, respectively. Total extracts were also Western blotted with IRS-1 and PI3K p85 as controls. Data are representative of at least three experiments IR, insulin receptor. D, immunopurified IRS-1 was labeled in vitro with UDP-[³H]GlcNAc by incubating with or without OGT, Sepharose A/G and washed, and then insulin receptor, stimulated or not with 1 μm insulin, was added for 20 min, and insulin-stimulated insulin receptor phosphorylation of Tyr(P)⁶⁰⁸ IRS-1 was measured by Western blot analysis using Tyr(P)⁶⁰⁸ IRS-1 antibody. Western blot intensity was quantitated using ImageJ.

FIGURE 4.

O-GlcNAc-modified IRS-1 does not interact with PI3K p85. PI3K p85 was immunoprecipitated (IP) from 3T3-L1 adipocytes and (A) probed (WB) for O-GlcNAc and then stripped and (B) reprobed for Tyr(P)⁶⁰⁸ (pY608) IRS-1 and for PI3K p85 (to demonstrate equivalent protein).

FIGURE 5.

PDK1 interacts with OGT upon insulin treatment, and elevated O-GlcNAc dampens this response. A, PDK was immunoprecipitated (IP) from 3T3-L1 adipocytes pretreated or not with PUGNAc, stimulated or not with 5 nm insulin, and then Western blotted (WB) for the presence of OGT and PDK1. See supplemental Fig. 2 for whole Western blot. B, total cell extracts were Western blotted for O-GlcNAc. C, immunoprecipitates of PDK1 pretreated or not with PUGNAc and stimulated or not with insulin were also Western blotted for O-GlcNAc and for PDK1. IgG immunoprecipitation was used as a negative control.

FIGURE 6.

Sites within the early steps of the insulin signaling pathway affected by OGT and O-GlcNAcylation. Upon insulin treatment of 3T3-L1 adipocytes, OGT is recruited to the insulin receptor (IR), tyrosine-phosphorylated, and catalytically activated (13) (1). OGT in turn modifies proteins, such as transcription factors (Sp1 and STAT3) (2) and kinases (PDK1), as well as a potential host of other unknown proteins (3). Acute insulin stimulation induces OGT interaction with at least PDK1 in the insulin signaling pathway and an increase in O-GlcNAc modification (4). Elevation of O-GlcNAc induced by PUGNAc inhibits OGT interaction with PDK1. Wortmannin (5) is a general inhibitor of PI3K, which blocks downstream AKT activation (6). During normal insulin-stimulated insulin receptor/AKT signaling, the insulin receptor becomes activated and phosphorylates the IRS-1 and IRS-2 YXXM motifs (Y⁶⁰⁸XXM and Y⁶²⁸XXM), which recruit and activate PI3K p85, resulting in downstream activation of PDK1, which in turn phosphorylates AKT (Thr³⁰⁸), resulting in GLUT-4 glucose uptake (7). Elevations in O-GlcNAc specifically induced by the O-GlcNAcase inhibitor PUGNAc result in O-GlcNAc modification of IRS-1 and decreased phosphorylation of Y⁶⁰⁸XXM (8), decreased PI3K p85 interaction with IRS-1, and downstream reduced phosphorylation of Thr³⁰⁸ of AKT, resulting in reduced GLUT-4 glucose uptake. IRS-1, IRS-2, and PDK1 were all excellent substrates for OGT in vitro (9). O-GlcNAc modification of PDK1 may play a role in reduced AKT activation.