Inhibition of O-GlcNAcase Using a Potent and Cell-Permeable Inhibitor Does Not Induce Insulin Resistance in 3T3-L1 Adipocytes

Summary To probe increased O-GlcNAc levels as an independent mechanism governing insulin resistance in 3T3-L1 adipocytes, a new class of O-GlcNAcase (OGA) inhibitor was studied. 6-Acetamido-6-deoxy-castanospermine (6-Ac-Cas) is a potent inhibitor of OGA. The structure of 6-Ac-Cas bound in the active site of an OGA homolog reveals structural features contributing to its potency. Treatment of 3T3-L1 adipocytes with 6-Ac-Cas increases O-GlcNAc levels in a dose-dependent manner. These increases in O-GlcNAc levels do not induce insulin resistance functionally, measured using a 2-deoxyglucose (2-DOG) uptake assay, or at the molecular level, determined by evaluating levels of phosphorylated IRS-1 and Akt. These results, and others described, provide a structural blueprint for improved inhibitors and collectively suggest that increased O-GlcNAc levels, brought about by inhibition of OGA, does not by itself cause insulin resistance in 3T3-L1 adipocytes.

The upper panel shows the power supplied to the system to maintain a constant temperature against time (the area of each peak gives the heat of interaction for that injection). The lower panel shows the bimolecular fit of the normalised heats of interaction plotted against the molar concentration. Assays were carried out in 50 mM MES, pH 6.5, 200 mM NaCl at 25 °C. The data of K d and ΔH a are average values from duplicate runs. (C) Three gels were run that were loaded identically to that in panel A. Proteins from these gels were transferred to nitrocellulose membrane using a wet protocol and probed for -actin using an anti--actin antibody, followed by goat anti-mouse IgG secondary antibody conjugated to horseradish peroxidase (HRP). Following washing and addition of the HRP substrate, the blots were exposed to film for different times as indicated. These three films were then scanned and analyzed by densitometry for quantitation.
(D) Densitometry of the band corresponding to -actin for each quantity of protein loaded in panel C. Each exposure length is plotted as different symbols; 5 seconds (blue diamonds); 20 seconds (green circles); and 2 minutes (red squares). Densitometric measurements are approximately linear in the range of 5 -50 g of total protein, however, linearity broke down faster at high protein amounts with a longer exposure time.
(E) Two gels were loaded identically to that in panel A and electrophoresed. Proteins from these gels were transferred to nitrocellulose membrane using a wet protocol and probed for O-GlcNAc using an anti-O-GlcNAc antibody (CTD110.6) followed, after washing, by goat anti-mouse IgM secondary antibody conjugated to horseradish peroxidase (HRP). Following addition of the HRP substrate, the blots were exposed to film to different times as indicated. These three films were then scanned and analyzed by densitometry for quantitation.
(F) Densitometry of the entire lane for each quantity of protein loaded in panel E. Each exposure length is plotted as different symbols: 5 seconds (blue diamonds); 20 seconds (green circles). Densitometric measurements are approximately linear in the range of 5 -50 g of total protein, however, linearity breaks down faster at low protein amounts with a shorter exposure time.
(G) A gel was loaded identically to that in panel A and electrophoresed. In the same manner as described above in E, the blot was probed for O-GlcNAc except this time a fluorescently labeled secondary antibody was used. In this case, a Typhoon imager was used for visualization and quantitation.

SUPPLEMENTAL EXPERIMENTAL PROCEDURES
BtGH84 Kinetics -Kinetic studies were conducted by monitoring the change in UV-Visible absorbance at 400 nm using a Cintra 10 spectrophotometer. Assays were carried out at 25°C in thermally equilibrated disposable cuvettes, containing a total volume of 1 ml buffer (50 mM MES, pH 6.5, 200 mM NaCl) with 50 μM 4-nitrophenyl 2-acetamido-2-deoxy--D-glucopyranoside (pNP-GlcNAc) as substrate. Reactions were initiated by the addition of 10 μL of 1.9 μM BtGH84 using a syringe. 6-Ac-Cas concentrations in the assay varied from 200 nM to 1 μM. 4-Nitrophenolate release was recorded continuously for 300 seconds. Rates in both the presence and absence of inhibitor were determined as the slope of the linear region over a 100 second period. The K i (inhibition constant) value was determined using the equation: where  o and  i are the rates of reaction in the absence and presence of inhibitor, respectively. A plot of v 0 /v i against increased inhibitor concentrations yields a gradient of 1/K i , with an intercept of 1.
Isothermal Titration Calorimetry (ITC) with BtGH84 -ITC measurements were performed using a MicroCal VC calorimeter (Northampton, MA). All measurements were carried out in 50 mM MES, pH 6.5 and 200 mM NaCl at 25°C. Purified BtGH84 protein was extensively dialyzed against the above buffer, and the dialyzate was subsequently used to dilute the 6-Ac-Cas. The enzyme and ligand concentrations used in these experiments were 34-50 μM and 0.5 mM, respectively. All samples were centrifuged and degassed prior to use. For each titration, 10 μL aliquots of the inhibitor were injected into BtGH84 in the cell, at an interval of 4 min, with 307 rpm stirring speed. The experimental data were fitted to a non-linear regression model using Microcal Origin software, with stoichiometry (n), enthalpy (ΔH°) and association constant (K a ) as adjustable parameters. The thermodynamic parameters ΔG° (free energy) and ΔS° (entropy) were derived from the standard equation: RT ln(K a ) = ΔG° = ΔH° − TΔS°.