Fig 2: Glucose concentration alters the expression of OGT isoforms and O-GlcNAc levels in mitochondria. (A) Schematic structure of OGT isoforms. (B) mOGT and ncOGT transcript levels were determined using RT-qPCR, and the results were referenced to HPRT1 and GAPDH housekeeping genes mRNA. The bars represent mean +/− SD from three independent experiments in duplicate. (C) O-GlcNAc, OGT, and OGA levels in cytoplasmic (30 μg of proteins per line) and mitochondrial (60 μg of proteins per line) fractions of breast cancer cells. For cytoplasmic extract, β-actin was used as a loading control, whereas cytochrome C was used as a marker of mitochondria-enriched fraction. * indicates significance; p < 0.05; ** p < 0.01; *** p < 0.001. Glc, glucose. (The original western blot images are shown in the File S1).

Fig 3: Impaired insulin secretion and processing in β-cell OGA deficient islets. β-cell mass of Control and βOGAKO female mice at 30-wks of age (A, n=3-4). In Vitro glucose stimulated insulin secretion assay with isolated primary islets from control and βOGAKO mice at 30-wks of age (B) with post-assay insulin content normalized to DNA (C) (n=5-6). Primary islet insulin (D) and proinsulin€) content normalized to DNA with proinsulin to insulin content ratio (F) (5-6). Immunofluorescence image of pancreatic islets showing CPE (red) and insulin (white) from Control and βOGAKO pancreas (G) and intensity quantification of CPE in insulin positive cells (H). Islet analysis from n=2-3 animals. 40x magnification. Scale bar = 50 μm. Representative western blot (I) and quantification of CPE (J) and eIF4G1 (K), normalized to beta-Actin (n=3-4). Statistical analyses were conducted using two-way ANOVA and unpaired, 2-way student t-test with significance *p<0.05, ** p<0.001, ****p<0.0001, ####pp<0.0001 relative to control fasting.

Fig 4: HRECs exposed to TNF-α, and high glucose for 24 h demonstrated increased total O-GlcNAcylation. O-GlcNAcylation confirmed by protein expression (clone: CTD.116.0) (A), protein expression of O-glycosyl transferase (OGT) and O-GlcNAcase (OGA) enzyme (B). HRECs treated with PUGNAc served as positive control. β-tubulin served as an internal control. Data represent Mean ± SEM from 3 independent experiments performed in duplicates. ***p < 0.001; **p < 0.01; *p < 0.05, #p > 0.05.

Fig 5: Loss of OGA in β-Cells Leads to Glucose Intolerance in Age-Dependent Manner in normal chow diet. In vivo glucose tolerance (2 g/kg glucose, i.p.) test of 8-wks (A, Male, n=7-12; B, Female, n=13-17) and insulin sensitivity (0.75 U/kg insulin, i.p.) of 10-wks (C, Male, n=8-10; D, Female, n=9-15). Glucose tolerance test of 26-wks (E, Male, n=7; F, Female, n=3-4) old mice and insulin of 28-wks old male (G, n=5-6) mice. In vivo glucose stimulated insulin secretion of male (H, n=5-6) with calculated stimulation index. Area under curve (AUC) of the blood glucose curves are presented for each figure. Statistical analyses were conducted using two-way ANOVA and unpaired, 2-way student t-test with significance *p < 0.05, #p<0.05 relative to fasting control. ns, non-statistical significance.