Changes in thymus size, cellularity and expression of CD4+ and CD8+ coreceptors induced by O-GlcNAcylation, through regulation of AMPK

Abstract

Introduction: Glycosylation with N-acetyl-glucosamine (O-GlcNAc) has been shown to be essential during T cell activation. Objective: The present study evaluated the impact of increased levels of O-GlcNAcylation on thymic size, cellularity and expression of CD4+ and CD8+ surface markers, through the regulation of adenosine monophosphate-activated protein kinase (AMPK) and caspase-3. Material and Methods: Male Wistar rats were treated with glucosamine 300 mg/kg or saline for 21 days. The thymus was collected for analysis of size, cellularity, morphometry and protein quantification by western blotting (O-GlcNAc, OGT, CD4+, CD8+, phospho-AMPK, caspase-3). Results: Glucosamine treatment increased global levels of O-GlcNAc [1.00 vs 2.14 ± 0.19 AU] and OGT [1.00 vs 1.31 ± 0.10 AU] in thymic tissue. O-glycosylation increased thymic index (0.10 vs 0.12 ± 0.00 kg/rat) and cellularity (446.90 ± 55.12 vs 891.10 ± 142.30 cells/mL), but was not able to promote morphological changes. The active form of AMPK was overexpressed (1.00 vs 1.48 ± 0.09 AU) and caspase-3 was reduced (1.0 vs 0.44 ± 0.07 AU). O-GlcNAc reduced the expression of CD4+ (1.00 vs 0.68 ± 0.08 AU) and CD8+ (1.00 vs 0.66 ± 0.10 AU) markers. Conclusion: O-GlcNAcylation induces an increase in thymic size and cellularity by inhibiting the caspase-3 apoptotic pathway through AMPK activation. However, O-GlcNAcylation reduces the expression of CD4+ and CD8+ coreceptors, demonstrating that O-GlcNAc may represent a novel mechanism for thymocyte modulation. Introduction: Glycosylation with N-acetyl-glucosamine (O-GlcNAc) has been shown to be essential during T cell activation. Objective: The present study evaluated the impact of increased levels of O-GlcNAcylation on thymic size, cellularity and expression of CD4+ and CD8+ surface markers, through the regulation of adenosine monophosphate-activated protein kinase (AMPK) and caspase-3. Material and Methods: Male Wistar rats were treated with glucosamine 300 mg/kg or saline for 21 days. The thymus was collected for analysis of size, cellularity, morphometry and protein quantification by western blotting (O-GlcNAc, OGT, CD4+, CD8+, phospho-AMPK, caspase-3). Results: Glucosamine treatment increased global levels of O-GlcNAc [1.00 vs 2.14 ± 0.19 AU] and OGT [1.00 vs 1.31 ± 0.10 AU] in thymic tissue. O-glycosylation increased thymic index (0.10 vs 0.12 ± 0.00 kg/rat) and cellularity (446.90 ± 55.12 vs 891.10 ± 142.30 cells/mL), but was not able to promote morphological changes. The active form of AMPK was overexpressed (1.00 vs 1.48 ± 0.09 AU) and caspase-3 was reduced (1.0 vs 0.44 ± 0.07 AU). O-GlcNAc reduced the expression of CD4+ (1.00 vs 0.68 ± 0.08 AU) and CD8+ (1.00 vs 0.66 ± 0.10 AU) markers. Conclusion: O-GlcNAcylation induces an increase in thymic size and cellularity by inhibiting the caspase-3 apoptotic pathway through AMPK activation. However, O-GlcNAcylation reduces the expression of CD4+ and CD8+ coreceptors, demonstrating that O-GlcNAc may represent a novel mechanism for thymocyte modulation.