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The Identification of a Novel Calcium-Dependent Link Between NAD⁺ and Glucose Deprivation-Induced Increases in Protein O-GlcNAcylation and ER Stress.

ABSTRACT: The modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is associated with the regulation of numerous cellular processes. Despite the importance of O-GlcNAc in mediating cellular function our understanding of the mechanisms that regulate O-GlcNAc levels is limited. One factor known to regulate protein O-GlcNAc levels is nutrient availability; however, the fact that nutrient deficient states such as ischemia increase O-GlcNAc levels suggests that other factors also contribute to regulating O-GlcNAc levels. We have previously reported that in unstressed cardiomyocytes exogenous NAD⁺ resulted in a time and dose dependent decrease in O-GlcNAc levels. Therefore, we postulated that NAD⁺ and cellular O-GlcNAc levels may be coordinately regulated. Using glucose deprivation as a model system in an immortalized human ventricular cell line, we examined the influence of extracellular NAD⁺ on cellular O-GlcNAc levels and ER stress in the presence and absence of glucose. We found that NAD⁺ completely blocked the increase in O-GlcNAc induced by glucose deprivation and suppressed the activation of ER stress. The NAD⁺ metabolite cyclic ADP-ribose (cADPR) had similar effects on O-GlcNAc and ER stress suggesting a common underlying mechanism. cADPR is a ryanodine receptor (RyR) agonist and like caffeine, which also activates the RyR, both mimicked the effects of NAD⁺. SERCA inhibition, which also reduces ER/SR Ca²⁺ levels had similar effects to both NAD⁺ and cADPR on O-GlcNAc and ER stress responses to glucose deprivation. The observation that NAD⁺, cADPR, and caffeine all attenuated the increase in O-GlcNAc and ER stress in response to glucose deprivation, suggests a potential common mechanism, linked to ER/SR Ca²⁺ levels, underlying their activation. Moreover, we showed that TRPM2, a plasma membrane cation channel was necessary for the cellular responses to glucose deprivation. Collectively, these findings support a novel Ca²⁺-dependent mechanism underlying glucose deprivation induced increase in O-GlcNAc and ER stress.

SUBMITTER: Zou L

PROVIDER: S-EPMC8691773 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:As an energy-sensitive post-translational modification, O-GlcNAcylation plays a major role in endothelial nitric oxide synthase (eNOS) activity regulation. However, effects of glucose deprivation on eNOS O-GlcNAcylation and the presence of novel O-GlcNAcylation sites of eNOS under glucose deprivation remain unknown. Hence, we aim to determine the effects of glucose deprivation on O-GlcNAcylation and novel O-GlcNAcylation sites of eNOS. Bovine aortic endothelial cells (BAECs) and Sprague-Dawley rats were induced by glucose deprivation and their eNOS O-GlcNAcylation was subjected to immunoblotting. eNOS and transfected eNOS were purified by pull-down assay and immunoprecipitation respectively. Novel O-GlcNAcylation sites of eNOS were predicted by HPLC-MS and MS/MS Ion and determined by immunoblotting. eNOS activity was detected by Elisa and isotope labeling method. In BAECs and rat thoracic aorta, low glucose-associated activation of eNOS was accompanied by elevated O-GlcNAcylation, which did not affect O-linked serine phosphorylation at 1179/1177 residues. Changes in this post-translational modification were associated with increased O-GlcNAc transferase (OGT) expression and were reversed by AMPK knockdown. Immunoblot analysis of cells expressing His-tagged wild-type human eNOS and human eNOS carrying a mutation at the Ser1177 phosphorylation site confirmed an increase in O-GlcNAcylation by glucose deprivation. A marked increase in O-GlcNAcylation indicated that eNOS contained novel O-GlcNAcylation sites that were activated by glucose deprivation. Immunoblot analysis of cells expressing His-tagged human eNOS carrying a mutation at Ser738 and Ser867 confirmed an increase in O-GlcNAcylation by glucose deprivation. Conversely, in His-tagged human eNOS carrying a mutation at Thr866, O-GlcNAcylation was unaffected by glucose deprivation. Differences in culture conditions were identified using two-way analysis of variance (ANOVA), one-way ANOVA, and unpaired Student's t-test. Glucose deprivation increases O-GlcNAcylation and activity of eNOS, potentially by the AMPK-OGT pathway, suggesting that Thr866 is a novel O-GlcNAcylation site involved in glucose-deprivation mediated eNOS activation.

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The Identification of a Novel Calcium-Dependent Link Between NAD⁺ and Glucose Deprivation-Induced Increases in Protein O-GlcNAcylation and ER Stress.

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The Identification of a Novel Calcium-Dependent Link Between NAD+ and Glucose Deprivation-Induced Increases in Protein O-GlcNAcylation and ER Stress.

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The Identification of a Novel Calcium-Dependent Link Between NAD⁺ and Glucose Deprivation-Induced Increases in Protein O-GlcNAcylation and ER Stress.