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Glucose deprivation-induced increase in protein O-GlcNAcylation in cardiomyocytes is calcium-dependent.

ABSTRACT: The posttranslational modification of nuclear and cytosolic proteins by O-linked ?-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such as neonatal cardiomyocytes, glucose deprivation increases O-GlcNAc levels and if so by what mechanism. Glucose deprivation significantly increased cardiomyocyte O-GlcNAc levels in a time-dependent manner and was associated with decreased O-GlcNAcase (OGA) but not O-GlcNAc transferase (OGT) protein. This response was unaffected by either the addition of pyruvate as an alternative energy source or by the p38 MAPK inhibitor SB203580. However, the response to glucose deprivation was blocked completely by glucosamine, but not by inhibition of OGA with 2-acetamido-2-deoxy-d-glucopyranosylidene)amino-N-phenylcarbamate. Interestingly, the CaMKII inhibitor KN93 also significantly reduced the response to glucose deprivation. Lowering extracellular Ca(2+) with EGTA or blocking store operated Ca(2+) entry with SKF96365 also attenuated the glucose deprivation-induced increase in O-GlcNAc. In C2C12 and HEK293 cells both glucose deprivation and heat shock increased O-GlcNAc levels, and CaMKII inhibitor KN93 attenuated the response to both stresses. These results suggest that increased intracellular calcium and subsequent activation of CaMKII play a key role in regulating the stress-induced increase in cellular O-GlcNAc levels.

SUBMITTER: Zou L

PROVIDER: S-EPMC3464547 | biostudies-literature | 2012 Oct

REPOSITORIES: biostudies-literature

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Glucose deprivation-induced increase in protein O-GlcNAcylation in cardiomyocytes is calcium-dependent.

Zou Luyun L Zhu-Mauldin Xiaoyuan X Marchase Richard B RB Paterson Andrew J AJ Liu Jian J Yang Qinglin Q Chatham John C JC

The Journal of biological chemistry 20120820 41

The posttranslational modification of nuclear and cytosolic proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) has been shown to play an important role in cellular response to stress. Although increases in O-GlcNAc levels have typically been thought to be substrate-driven, studies in several transformed cell lines reported that glucose deprivation increased O-GlcNAc levels by a number of different mechanisms. A major goal of this study therefore was to determine whether in primary cells, such ...[more]

PMID: 22908225

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Project description: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.

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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Glucose deprivation-induced increase in protein O-GlcNAcylation in cardiomyocytes is calcium-dependent.

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Glucose deprivation-induced increase in protein O-GlcNAcylation in cardiomyocytes is calcium-dependent.

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