No abstract

Increased levels of protein O-linked N-acetylglucosamine (O-GlcNAc) have been shown to increase cell survival following stress. Therefore, the goal of this study was to determine whether in isolated neonatal rat ventricular myocytes (NRVMs) an increase in protein O-GlcNAcylation resulted in improved survival and viability following ischemia-reperfusion (I/R). NRVMs were exposed to 4 h of ischemia and 16 h of reperfusion, and cell viability, necrosis, apoptosis, and O-GlcNAc levels were assessed. Treatment of cells with glucosamine, hyperglycemia, or O-(2-acetamido-2-deoxy-D-glucopyranosylidene)-amino-N-phenylcarbamate(PUGNAc), an inhibitor of O-GlcNAcase, significantly increased O-GlcNAc levels and improved cell viability, as well as reducing both necrosis and apoptosis compared with untreated cells following I/R. Alloxan, an inhibitor of O-GlcNAc transferase, markedly reduced O-GlcNAc levels and exacerbated I/R injury. The improved survival with hyperglycemia was attenuated by azaserine, which inhibits glucose metabolism via the hexosamine biosynthesis pathway. Reperfusion in the absence of glucose reduced O-GlcNAc levels on reperfusion compared with normal glucose conditions and decreased cell viability. O-GlcNAc levels significantly correlated with cell viability during reperfusion. The effects of glucosamine and PUGNAc on cellular viability were associated with reduced calcineurin activation as measured by translocation of nuclear factor of activated T cells, suggesting that increased O-GlcNAc levels may attenuate I/R induced increase in cytosolic Ca(2+). These data support the concept that activation of metabolic pathways leading to an increase in O-GlcNAc levels is an endogenous stress-activated response and that augmentation of this response improves cell survival. Thus strategies designed to activate these pathways may represent novel interventions for inducing cardioprotection.

We have previously reported that glucosamine protected neonatal rat ventricular myocytes against ischemia-reperfusion (I/R) injury, and this was associated with an increase in protein O-linked-N-acetylglucosamine (O-GlcNAc) levels. However, the protective effect of glucosamine could be mediated via pathways other that O-GlcNAc formation; thus the initial goal of the present study was to determine whether increasing O-GlcNAc transferase (OGT) expression, which catalyzes the formation of O-GlcNAc, had a protective effect similar to that of glucosamine. To better understand the potential mechanism underlying O-GlcNAc-mediated cytoprotection, we examined whether increased O-GlcNAc levels altered the expression and translocation of members of the Bcl-2 protein family. Both glucosamine (5 mM) and OGT overexpression increased basal and I/R-induced O-GlcNAc levels, significantly decreased cellular injury, and attenuated loss of cytochrome c. Both interventions also attenuated the loss of mitochondrial membrane potential induced by H2O2 and were also associated with an increase in mitochondrial Bcl-2 levels but had no effect on Bad or Bax levels. Compared with glucosamine and OGT overexpression, NButGT (100 microM), an inhibitor of O-GlcNAcase, was less protective against I/R and H2O2 and did not affect Bcl-2 expression, despite a 5- to 10-fold greater increase in overall O-GlcNAc levels. Decreased OGT expression resulted in lower basal O-GlcNAc levels, prevented the I/R-induced increase in O-GlcNAc and mitochondrial Bcl-2, and increased cellular injury. These results demonstrate that the protective effects of glucosamine are mediated via increased formation of O-GlcNAc and suggest that this is due, in part, to enhanced mitochondrial Bcl-2 translocation.

([Ca 2ϩ ] i ) in cardiomyocytes and to have positive inotropic effects on the heart (1, 2). In addition to their importance in the acute regulation of cardiac function, their significance has increased further as the transcriptional pathways that lead to cardiac hypertrophy have been elucidated. Although initially compensatory, prolonged hypertrophy is often associated with decompensation, dilated cardiomyopathy, arrhythmia, fibrotic disease, and heart failure (3).The importance of agonists that activate PLC to cardiac hypertrophy is now well established (4). One well studied mouse model of cardiac hypertrophy involves the overexpression of a constitutively active form of a G protein subunit, G␣ q , which leads to chronic activation of PLC and the continuous production of IP 3 and diacylglycerol (5). In addition, overexpression in the heart of the PLC-activating angiotensin II (Ang II) type I receptor also leads to hypertrophy (6, 7). More clinically relevant, hypertrophied hearts induced by volume overload are commonly characterized by high levels of IP 3 -generating agonists such as Ang II (8).There are multiple signaling pathways downstream of PLC leading to cardiac hypertrophy. One involves diacylglycerol, protein kinase C, small guanine nucleotide-binding proteins (9), the MEK1-ERK1/2 branch of the mitogen-activated protein kinase pathway (10), and the transcription factor GATA4 (11, 12). Two others involve IP 3 and elevated levels of [Ca 2ϩ ] i . One of these is dependent on Ca 2ϩ /calmodulin-dependent calmodulin kinase and the transcription factor MEF2 (13), and the other is mediated by the Ca 2ϩ /calmodulin-activated protein phosphatase calcineurin and the transcription factor NFAT3 (14). The latter signaling pathway was first defined in lymphocytes (15) and is fundamental to an array of biological responses in a variety of cell types (16,17). A rise in [Ca 2ϩ ] i triggered by ligands generating IP 3 leads to the activation of the phosphatase activity of calcineurin, the dephosphorylation of NFAT family members, and their translocation to the nucleus to initiate transcription. Rapid export of NFAT from the nucleus when [Ca 2ϩ ] i levels drop prevents brief [Ca 2ϩ ] i pulses from initiating transcription of NFATdependent genes (15,16).A critical, unresolved issue for cardiac hypertrophy is the mechanism leading from IP 3 -mediated stimuli to elevated [Ca 2ϩ ] i . In most cell types, the initial increase in [Ca 2ϩ ] i in response to IP 3 -generating agonists is due to the release of Ca 2ϩ from the endoplasmic reticulum (ER

We have previously demonstrated that in a rat model of trauma-hemorrhage (T-H), glucosamine administration during resuscitation improved cardiac function, reduced circulating levels of inflammatory cytokines, and increased tissue levels of O-linked N-acetylglucosamine (O-GlcNAc) on proteins. The mechanism(s) by which glucosamine mediated its protective effect were not determined; therefore, the goal of this study was to test the hypothesis that glucosamine treatment attenuated the activation of the nuclear factor-kappaB (NF-kappaB) signaling pathway in the heart via an increase in protein O-GlcNAc levels. Fasted male rats were subjected to T-H by bleeding to a mean arterial blood pressure of 40 mmHg for 90 min followed by resuscitation. Glucosamine treatment during resuscitation significantly attenuated the T-H-induced increase in cardiac levels of TNF-alpha and IL-6 mRNA, IkappaB-alpha phosphorylation, NF-kappaB, NF-kappaB DNA binding activity, ICAM-1, and MPO activity. LPS (2 microg/ml) increased the levels of IkappaB-alpha phosphorylation, TNF-alpha, ICAM-1, and NF-kappaB in primary cultured cardiomyocytes, which was significantly attenuated by glucosamine treatment and overexpression of O-GlcNAc transferase; both interventions also significantly increased O-GlcNAc levels. In contrast, the transfection of neonatal rat ventricular myocytes with OGT small-interfering RNA decreased O-GlcNAc transferase and O-GlcNAc levels and enhanced the LPS-induced increase in IkappaB-alpha phosphorylation. Glucosamine treatment of macrophage cell line RAW 264.7 also increased O-GlcNAc levels and attenuated the LPS-induced activation of NF-kappaB. These results demonstrate that the modulation of O-GlcNAc levels alters the response of cardiomyocytes to the activation of the NF-kappaB pathway, which may contribute to the glucosamine-mediated improvement in cardiac function following hemorrhagic shock.

We report that acutely increasing O-GlcNAcylation in Sprague Dawley rat hippocampal slices induces an NMDA receptor and protein kinase C-independent long-term depression (LTD) at hippocampal CA3-CA1 synapses (O-GcNAc LTD). This LTD requires AMPAR GluA2 subunits, which we demonstrate are O-GlcNAcylated. Increasing O-GlcNAcylation interferes with long-term potentiation, and in hippocampal behavioral assays, it prevents novel object recognition and placement without affecting contextual fear conditioning. Our findings provide evidence that O-GlcNAcylation dynamically modulates hippocampal synaptic function and learning and memory, and suggest that altered O-GlcNAc levels could underlie cognitive dysfunction in neurological diseases.

The hypothesis that intercellular adhesion can be subdivided into two separable phenomena-an initial recognition event and a subsequent stabilization-is supported by the use of a cell binding assay that provides a quantitative measure of intercellular binding strengths. Radioactive single cells are brought into contact with cell monolayers at 40C in sealed compartments. The compartments are inverted and a centrifugal force is then applied to dislodge the probe cells from the monolayers. By varying the speed of centrifugation, the force maintaining associations between embryonic chicken neural retina cells was determined to be on the order of 10-5 dyne. Topographic specificities of single neural retina cells for retinal monolayers from various regions of the retina were detected with this assay and corresponded to those observed in more traditional assays at 37C. Also observed were two time-and temperature-dependent stabilization processes in which the force required for dislodgment increased. One of the stabilization processes was sensitive to dinitrophenol and was inactive at 4TC; the second was still active in metabolically blocked cells. The metabolic-dependent process resulted in interactions at least 13 times as strong as the initial binding. The metabolic-independent process resulted in about a 2-fold increase in binding strength and had a temperature dependence similar to that of membrane diffusional phenomena.Precise cellular interactions play an essential role in the assembly of tissues during embryogenesis and are attributed, in part, to adhesive specificities (1). Studies by Umbreit and Roseman (2) on embryonic chicken tissues in vitro suggested that cell adhesion could be subdivided into an initial reversible phase and a subsequent stabilizing process. Reversible adhesions were characterized by their dissociation by small fluid shear forces. The stabilizing process was strongly dependent on time and temperature and led to more permanent bonds between cells.Data obtained by using a newly developed cell binding assay support these results and suggest that interactions determining recognition specificity take place during the reversible phase ofcell interactions. In addition, the assay provides a quantitative measure of intercellular binding strengths. Radioactive probe cells are brought into contact with monolayers in sealed compartments by a low centrifugal force. The compartments are then inverted in the centrifuge and the force required to dislodge the probe cells from the monolayers is determined. Probe cells were found to bind at 4°C with a strength that resisted about 10' dyne of dislodgment force per cell and exhibited recognition specificity and sensitivity to trypsin and calcium. When the bound cells were incubated for 8 min at 37°C, the resistance to dislodgment was at least 13 times stronger than that of the associations formed at 4°C. This stabilization was dependent on the cells' being metabolically active.With metabolism inhibited, a second type of strengthening ofcell binding wa...

We have previously shown that preischemic treatment with glucosamine improved cardiac functional recovery following ischemia-reperfusion, and this was mediated, at least in part, via enhanced flux through the hexosamine biosynthesis pathway and subsequently elevated O-linked N-acetylglucosamine (O-GlcNAc) protein levels. However, preischemic treatment is typically impractical in a clinical setting; therefore, the goal of this study was to investigate whether increasing protein O-GlcNAc levels only during reperfusion also improved recovery. Isolated perfused rat hearts were subjected to 20 min of global, no-flow ischemia followed by 60 min of reperfusion. Administration of glucosamine (10 mM) or an inhibitor of O-GlcNAcase, O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 200 microM), during the first 20 min of reperfusion significantly improved cardiac functional recovery and reduced troponin release during reperfusion compared with untreated control. Both interventions also significantly increased the levels of protein O-GlcNAc and ATP levels. We also found that both glucosamine and PUGNAc attenuated calpain-mediated proteolysis of alpha-fodrin as well as Ca(2+)/calmodulin-dependent protein kinase II during reperfusion. Thus two independent strategies for increasing protein O-GlcNAc levels in the heart during reperfusion significantly improved recovery, and this was correlated with attenuation of calcium-mediated proteolysis. These data provide further support for the concept that increasing cardiac O-GlcNAc levels may be a clinically relevant cardioprotective strategy and suggest that this protection could be due, at least in part, to inhibition of calcium-mediated stress responses.