Fig 2: OGT is required for the neuronal activity of the VMH.(A) Basal membrane potential of SF1 neurons in CTL and VOK mice. (B) Firing rate of SF1 neurons in CTL and VOK mice. (C) Representative traces of action potentials of SF1 neurons in CTL and VOK mice. (D) The role of OGT in VMH neurons to regulate energy balance and white adipose tissue lipolysis. SF1 neurons from 5- to 7-week-old age-matched male CTL and VOK mice fed ad libitum on a normal chow diet were collected for electrophysiology. CTL SF1 neurons: n = 26 and VOK SF1 neurons: n = 20. Data are shown as means ± SEM. ***P < 0.001 by unpaired Student’s t test.

Fig 3: OGT expression and activity was upregulated in UCB cell models with acquired chemoresistance to gemcitabine (GEM) and paclitaxel (PTX). (A) Cell morphology of resistant and nonresistant cells observed under a light microscope (magnification, ×10). UMUC-3 and UMUC-3-GEM-resistant (GEMr) cells were treated with a vehicle or 10 μM GEM. T24 and T24-PTX-resistant (PTXr) cells were treated with a vehicle or 1 μM PTX. Observations were made 72 h after treatment. After observation, cell viability was analyzed by MTT assay. The data represent at least three independent experiments and error bars indicate the mean ± SEM. (B) qRT-PCR was used to detect OGT and GAPDH expression in UMUC-3/UMUC-3-GEMr and T24/T24-PTXr cells. Error bars indicate the mean ± SEM for three independent regions each. (C) Immunoblots of lysates derived from UMUC-3, UMUC-3-GEMr, T24, and T24-PTXr cells against OGT and O-GlcNAc. β-actin was used as the loading control. Error bars indicate the mean ± SEM for three independent regions each. For all panels * p < 0.05; ** p < 0.01; *** p < 0.001.

Fig 4: RANKL-induced p65 and NFATc1 O-GlcNAcylation are required for translocation to the nucleus. (A,B) BMMs were incubated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the absence or presence of 5 μM OSMI-1 for 5 min. Western blot analysis of O-GlcNAc, Ogt, or p65 was performed after immunoprecipitation using O-GlcNAc or Ogt antibodies (right panel). The immunoprecipitation band density was quantified (left panel). (C,D) BMMs were incubated with M-CSF (30 ng/mL) and RANKL (100 ng/mL) in the absence or presence of 5 μM OSMI-1 for 48 h. Western blot analysis of O-GlcNAc, Ogt, or NFATc1 was performed after immunoprecipitation using O-GlcNAc or Ogt antibodies (right panel). The immunoprecipitation band density was quantified (left panel). (E) Cytosolic and nuclear proteins were extracted for BMMs in the absence or presence of 5 μM OSMI-1 for 5 min to determine translocation of p-p65 (right panel). The band density was quantified (left panel). (F) Cytosolic and nuclear proteins were extracted for BMM in the absence or presence of 5 μM OSMI-1 for 48 h to determine translocation of NFATc1 (right panel). The band density was quantified (left panel). Statistical data are presented as the means ± S.E.M. (** p < 0.01 and *** p < 0.001, as measured by Student’s t-test).

Fig 5: Working model for inhibition of retinal protein O-GlcNAcylation by Ang1-7. The results support a model wherein Ang1-7 inhibits O-GlcNAcylation-induced reactive oxygen species (ROS) via a novel Mas/EPAC/Rap1/OGT signaling axis. Ang1-7 activates the Mas receptor to enhance cAMP levels and activate EPAC. In turn, EPAC promotes Rap1 GDP release and GTP binding. Activation of this signaling axis suppresses protein O-GlcNAcylation downstream of GFAT by inhibiting OGT activity.