Elevated nucleocytoplasmic glycosylation by O-GlcNAc results in insulin resistance associated with defects in Akt activation in 3T3-L1 adipocytes

Abstract

Increased flux of glucose through the hexosamine biosynthetic pathway (HSP) is believed to mediate hyperglycemia-induced insulin resistance in diabetes. The end product of the HSP, UDP beta-N-acetylglucosamine (GlcNAc), is a donor sugar nucleotide for complex glycosylation in the secretory pathway and for O-linked GlcNAc (O-GlcNAc) addition to nucleocytoplasmic proteins. Cycling of the O-GlcNAc posttranslational modification was blocked by pharmacological inhibition of O-GlcNAcase, the enzyme that catalyzes O-GlcNAc removal from proteins, with O-(2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc). PUGNAc treatment increased levels of O-GlcNAc and caused insulin resistance in 3T3-L1 adipocytes. Insulin resistance induced through the HSP by glucosamine and chronic insulin treatment correlated with increased O-GlcNAc levels on nucleocytoplasmic proteins. Whereas insulin receptor autophosphorylation and insulin receptor substrate 2 tyrosine phosphorylation were not affected by PUGNAc inhibition of O-GlcNAcase, downstream phosphorylation of Akt at Thr-308 and glycogen synthase kinase 3 beta at Ser-9 was inhibited. PUGNAc-induced insulin resistance was associated with increased O-GlcNAc modification of several proteins including insulin receptor substrate 1 and beta-catenin, two important effectors of insulin signaling. These results suggest that elevation of O-GlcNAc levels attenuate insulin signaling and contribute to the mechanism by which increased flux through the HSP leads to insulin resistance in adipocytes.

Figure 1

Elevated levels of O-GlcNAc in 3T3-L1 adipocytes is linked to insulin resistance. 3T3-L1 adipocytes in the absence or presence of PUGNAc (100 μM) were (A) stimulated with 0, 1, or 10 nM insulin for 10 min and 50 μg of whole-cell lysate per treatment was immunoblotted with a general anti-O-GlcNAc antibody (110.6) or (B) stimulated with various concentrations of insulin for 10 min and [¹⁴C]-2-DOG uptake was measured and plotted as insulin-dependent uptake. The percent resistance induced by PUGNAc at each insulin concentration is given numerically above the bar graph. (C) 3T3-L1 adipocytes in the presence of indicated concentrations of glucosamine and/or insulin were analyzed for [¹⁴C]-2-DOG uptake during a 10-min insulin stimulation (10 nM) or whole-cell lysates were immunoblotted with the 110.6 antibody.

Figure 2

PUGNAc does not affect insulin receptor or IRS protein tyrosine phosphorylation or GLUT4 levels. 3T3-L1 adipocytes were starved of growth factors in the absence or presence of PUGNAc (100 μM) and were acutely treated with 10 nM insulin for various times. (A) Whole-cell lysates were immunoblotted with antiphosphotyrosine (PY20) and positions of the insulin receptor β (IR) at 86 kDa and IRS proteins (IRS-1/2) at ≈180 kDa are indicated. (B) Insulin receptor (IR) β immunoprecipitates were immunoblotted with antibodies to either insulin receptor β or phosphotyrosine. (C) IRS-2 immuno-precipitates were immunoblotted with antibodies to either IRS-2 or phosphotyrosine. (D) Whole-cell lysates were immunoblotted with anti-GLUT 4 antibody. WB:PY, phosphotyrosine Western blot; IP, immunoprecipitation.

Figure 3

PUGNAc-induced elevation of O-GlcNAc inhibits insulin-stimulated Akt phosphorylation at Thr-308. 3T3-L1 adipocytes were starved of growth factors in the absence or presence of PUGNAc (100 μM) and acutely stimulated with 10 nM insulin for 5 min. Various amounts of whole-cell lysates were immunoblotted with antibodies recognizing either (A) Akt or (B) phosphorylated Thr-308–Akt. (C) Densitometry of bands from phosphorylated Thr-308–Akt immunoblot.

Figure 4

Insulin-stimulated phosphorylation of GSK3β is reduced in PUGNAc-treated cells. 3T3-L1 adipocytes were starved of growth factors in the absence or presence of PUGNAc (100 μM) and stimulated with insulin at concentrations of 1 nM or 10 nM for 5 min. Whole-cell lysates were separated by SDS/PAGE and immunoblotted with either (A) anti-GSK3β or (B) an antibody recognizing GSK3β phosphorylated at Ser-9. (C) Densitometry of bands from phosphorylated Ser-9–GSK3β Western blot.

Figure 5

O-GlcNAc modification of IRS-1 and β-catenin increases in insulin-resistant 3T3-L1 adipocytes. 3T3-L1 adipocytes were starved of growth factors in the absence or presence of PUGNAc (100 μM). (A) Whole-cell lysates were immunoprecipitated with the general O-GlcNAc antibody 110.6 or anti-IgM agarose (control), separated by SDS/PAGE, and immunoblotted with an antibody to IRS-1. (B) Whole-cell lysates were immunoprecipitated with anti-Akt or (*) anti-Akt antibody in the absence of lysate as a negative control, separated by SDS/PAGE, and immunoblotted with anti-Akt or the general O-GlcNAc antibody 110.6. (C) Whole-cell lysates were immunoprecipitated with anti-GSK3β, separated by SDS/PAGE, and immunoblotted with anti-GSK3β or the general O-GlcNAc antibody 110.6. (D) Whole-cell lysates were immuoprecipitated with anti-β-catenin, separated by SDS/PAGE, and immunoblotted with anti-β-catenin or the general O-GlcNAc antibody 110.6. IP, immunoprecipitation; WB, Western blot.