Activation of OGT prevents AMPK-inhibition induced 26S proteasome activation.

<p>AMPK suppression by compound C (10 µmol/L for 2 hour) in HUVEC (A) increases the association of PA700/S10B (from 19S complex) with β7 (from 20S complex) accompanied by a decrease of PA700/S10B O-GlcNAc modification, and (B) increases 26S proteasome activity, which can be prevented by pre-incubation of UDP-GlcNAc (25 µmol/L for 30 min), but not by PUGNAc (14 µmol/L for 30 min), the inhibitor of O-GlcNAcase. The blots shown are representative of 3 independent experiments.</p

AMPKα1 deficiency mildly increased clinical arthritis in K/BxN arthritis.

<p>WT mice and AMPKα1 KO were injected with 100μl serum from adult K/BxN mice on day 0 to induce arthritis. (A) Clinical score and ankle thickness in WT and AMPKα1 deficient animals (n = 5 mice per group) at day 6 after arthritis induction. (B) Proteins of joint tissues from naive (n = 4 mice per group) and arthritic mice (n = 4 mice per group) of WT and AMPKα1 deficient mice were extracted at day 6 after arthritis induction and analyzed by ELISA for the presence of IL-6. (C) Clinical score in WT animals (black circles, n = 6) and AMPKα1 animals (black squares, n = 6). Values are means ± SEM. * = p<0.05; ** = p<0.01.</p

A-769662 treatment abrogated joint damage and IL-6 expression in passive K/BxN arthritis.

<p>(A) Representative H&E and Safranin O stained sections of ankle joints on day 7 of arthritis induction in DMSO and A-769662-treated mice. Magnification 200x original. Black arrows in H&E stained sections show joint inflammation and white arrows in Safranin O stained sections show cartilage damage that was reduced in A-769662-treated ankles. (B) Histological scores for joint inflammation, erosion and cartilage damage in vehicle and high dose A-769662-treated mice on day 7 after serum transfer. (C) Proteins of joint tissues from naive (n = 3 mice per group) and arthritic mice (n = 5 mice per group) of DMSO and A-769662-treated mice were extracted and analyzed by ELISA for the presence of the indicated cytokines.</p

AMPK inhibited IL-6 and NO production in BMDMs stimulated with TLR2 and TLR4 agonists.

<p>(A,B) BMDMs were pre-treated with A-769662 (100, 250 or 500 μM) for 2 hours before stimulated with LPS (1 μg/ml) and Pam3Cys4 (1 μg/ml) for 18 hours. The conditioned media was subjected to ELISA and Griess reaction for the indicated cytokine and NO release, respectively. (C,D) BMDMs were pre-treated with A-769662 (100 μM) for 2 hours before stimulated with LPS for two hours. RNA was isolated and analyzed for the expression of the indicated genes. (E,F) BMDMs were pre-treated with A-769662 (100 μM) for 2 hours before stimulated with LPS (1 μg/ml) and Pam3Cys4 (1 μg/ml) for 18 hours. The conditioned media was subjected to ELISA for the indicated cytokines. Results are expressed as means ± SEM.* p< 0.05 vs DMSO; ** p<0.01; *** p<0.001 and are representative of four independent experiments.</p

Loss of OGT increases 26S proteasome assembly.

<p>HUVEC transfected with OGT siRNA but not control siRNA show an increase in (A) association of PA700/S10B (from 19S complex) with β7 (from 20S complex) and (B) 26S proteasome activity, mimicking the effect of AMPK-suppression by compound C (10 µmol/L for 2 hour). The blots shown are representative of 3 independent experiments. * represents p<0.05 vs the control (without compound C treatment).</p

A-769662 treatment also abrogated joint damage and IL-6 expression in AIA.

<p><b>(</b>A) Representative H&E and Safranin O stained sections of knee joints on day 10 of AIA induction in vehicle and A-769662-treated mice, which were treated either at day 0 or day 4 after intraarticular injection of mBSA (n = 5 mice per group). Original magnification 200x. (B) Knee thickness in A-769662-treated mice on day 10 of AIA induction. (C) Sections of knee joints were scored for inflammatory infiltration, bone erosion and cartilage damage. A-769662-treated mice had significantly lower scores than WT. (D) Proteins of joint tissues from naive (n = 3 mice per group) and arthritic mice (n = 5 mice per group) of vehicle and A-769662-treated mice were extracted and analyzed by ELISA for the presence of IL-6. (E) Serum from naive (n = 3 mice per group) and arthritic mice (n = 5 mice per group) of vehicle and A-769662-treated mice was analyzed by ELISA for IL-6. Results are expressed as means ± SEM.* p< 0.05 vs WT mice; ** p<0.01</p

A-769662 activated AMPKα and regulated NF-κB and MAPK signaling in BMDMs.

<p>BMDMs were cultured in the presence of different concentrations of A-769662 for 2 hours (A), or BMDMs were pre-treated with A-769662 for 2 hours before stimulated with LPS (1 μg/ml) (B). Lysates of BMDMs were prepared and analyzed for the expression of the indicated proteins by Western blot (WB). Semi-quantitative densitometry analysis of Western blots (arbitrary densitometry units) for phosphorylation of indicated proteins normalized to total protein is shown. Results are representative of three independent experiments.</p

Increased 26S proteasome activity in AMPK-suppressed HUVEC is correlated with the enhanced association of 19S and 20S sub-complexes.

<p>Compound C (10 µmol/L for 2 hour)-treated HUVEC present (A) AMPK inactivation, (B) an increase in association of PA700/S10B (from 19S complex) with β7 (from 20S complex), which can be blocked by AICAR pre-incubation (2 mmol/L for 6 hours), (C) 26S proteasome assembly (same samples were run on 3–14% native-PAGE under non-reducing condition followed by conventional Western blot on duplicated blots with PA700/S10B and β7 antibodies, respectively), and (D) an increase in 26S proteasome activity (chymotrypsin-like) (n = 3). The increased association of proteasome sub-complex is also observed in (E) HUVEC overexpressing AMPK-DN but not AMPK-CA or GFP. All of the blots shown are representative of 3 independent experiments. NS represents not significant.</p

AMPK depletion is associated with decreased association of OGT with proteasome and increased 26S proteasome assembly and activity in AMPK-KO mice.

<p>Gender (male) and age (12 weeks) matched mice (n = 8/group) with the genotypes of wild type (C57BL/6J) and AMPKα knockout were used. Compared to aortas from wild type (C57BL/6J) mice, (A) aortas from AMPKα knockout mice exhibit (B) a decrease in the association of OGT with proteasome (PA700/S10B), (C) an increase in proteasome assembly (PA700/S10B-β7 association), and (D) an increase in 26S proteasome activity, without alteration in the expression levels of proteasome (β7 or PA700/S10B) or OGT. * represents p<0.05 vs wild type (n = 8).</p

AMPK suppression increases 26S activity in HUVEC.

<p>AMPK suppression in HUVEC either by (A) compound C (10 µmol/L for 2 hour) or by (B) overexpression of AMPK-dominant negative mutant (DN) increases 26S proteasome activities, as demonstrated by ATP-dependent increased chymotrypsin-like, trypsin-like and caspase-like activity on fluorescent proteasome substrates. MG132 (0.5 µmol/L for 1 hour) or AICAR (2 mmol/L for 6 hours) treatment blocks the increased chymotrypsin-like activity in AMPK-suppressed HUVEC. * indicates P<0.05 vs vehicle (DMSO) or GFP, n = 3 per group.</p