Fig 2: Mice with SF1 neuron-specific OGT deletion show decreased energy expenditure and reduced lipolytic activity of white adipose tissues.(A to D) Metabolic cage analysis of food intake (A), physical activity (B), energy expenditure (C), and RER (D) of CTL and VOK mice. (E) Hematoxylin and eosin (H&E) staining of BAT, scWAT, pgWAT, and rpWAT of CTL and VOK mice after 6 hours of fasting. Scale bar, 50 μm. (F) Adipocyte sizes in different white adipose tissues of CTL and VOK mice. Individual dots represent individual adipocytes from three CTL and three VOK mice. (G) Western blot of p-HSLS563, HSL, and adipose triglyceride lipase (ATGL) in scWAT and pgWAT of CTL and VOK mice after 6 hours of fasting. Densitometric quantifications for the ratio of p-HSLS563 to total HSL are shown below. (H) Western blot of p-PKA substrate in scWAT of CTL and VOK mice after 6 hours of fasting. Densitometric quantification is shown on the right. CTL: n = 7 and VOK: n = 7 for the quantifications of the metabolic cage analysis. CTL: n = 3 and VOK: n = 5 for the representative traces of the metabolic cage analysis. Mice were aged 19 to 20 weeks before the body weight divergence. Data are shown as means ± SEM. *P < 0.05, **P < 0.01, and ****P < 0.0001 by unpaired Student’s t test. ns, not significant.

Fig 3: EGF increases PKM2 association with OGT.A, EGF increases the interaction between PKM2 and OGT. (top) A549 cells treated by EGF (100 ng/ml for 1 h) were prepared for IP with PKM2 or OGT antibody and analyzed by WB with indicated antibodies. (bottom) Relative protein levels of IP. OGT, OGA, and PKM2 O-GlcNAc were normalized to immunoprecipitated PKM2, while OGT pTyr and PKM2 were normalized to OGT. Quantification shows mean ± SD (n = 3) with significance determined by student’s t test, ∗∗∗p < 0.001, ns, nonsignificant. B, activated EGFR increases PKM2 binding to OGT. (top) In A549 cells deprived for serum and transfected with EGFR or EGFRvIII for 24 h, PKM2- or OGT-associated proteins were obtained from IP and examined by WB with indicated antibodies. (bottom) Relative protein levels of IP. OGT, OGA, PKM2 O-GlcNAc, OGT pTyr, and PKM2 were normalized to PKM2 or OGT. Quantification shows mean ± SD (n = 3) with significance determined by one-way ANOVA, ∗∗∗p < 0.001, ns, nonsignificant. C, assay for UDP-GlcNAc level. A549 cells were treated with EGF (100 ng/ml) for different durations and subjected to UDP-GlcNAc analysis by HPLC. Data represent mean ± SD (n = 3) with significance determined by one-way ANOVA, ns, nonsignificant. EGF, epidermal growth factor; EGFR, EGF receptor; OGA, O-GlcNAcase; OGT, O-GlcNAc transferase.

Fig 4: Networks for variables with significant correlations with O-GlcNAc related variables. A Male saline, B Male Thiamet G, C Female Saline, D Female Thiamet G. Kendall’s rank correlation tests were performed. All correlations are significant with Prob >|τ|< 0.05. Network visualization was performed using Cytoscape 3.8.2. The nodes are grouped based on the functions of the proteins or the localizations or the processes involved. Red edges denote positive relationships, whereas blue edges denote negative relationships. The thickness of the edges represents the strength of the relationships based on the Kendall Tau coefficient magnitude (Additional file 3: Tables S2–S5)

Fig 5: OGA activity is decreased in mice exposed to Thiamet G (TG). A OGA activity assay in cortical homogenates from male and female mice treated with either vehicle (0.9% saline; Sal) or TG. The rate of 4MU release over time was analyzed by two-way ANOVA and sex differences assessed by Student’s t-tests and false discovery rate controlled using the Benjamini–Hochberg method (q* = 0.05). ***p < 0.0001. B Representative image and western blot analysis of O-GlcNAcylated protein levels in saline-treated females (FS), TG-treated females (FT), saline-treated males (MS), and TG-treated males (MT) measured with the CTD110.6 antibody. ***p < 0.001, and ****p < 0.0001 compared to saline controls by student t-test (2-way ANOVA and post-hoc Tukey). C, D Representative image and western blot analyses of OGT and OGA protein levels