Elevated O-GlcNAc Levels Research Articles

GFPT1 accelerates immune escape in breast cancer by modifying PD-L1 via O-glycosylation

BackgroundImmune escape is one of the causes of poor prognosis in breast cancer (BC). Glutamine-fructose-6-phosphate transaminase 1 (GFPT1) is the first speed-limiting enzyme of the hexosamine biosynthesis pathway (HBP) and is essential for the progression of BC. Nevertheless, the mechanism of the influence of GFPT1 in BC immune escape is not clear.MethodsFirst, the level of GFPT1 in BC was analyzed by starbase, and GFPT1 expression in BC tissues was measured by qRT-PCR, western blot and IHC. Then, the O-GlcNAc levels were detected by western blot. Thereafter, Co-IP was applied to examine the relationship between GFPT1 and PD-L1. At last, a mouse model was constructed for validation in vivo.ResultsFirstly, we discovered that GFPT1 was obviously strengthened in BC. Knockdown or introduction of GFPT1 correspondingly degraded and elevated O-GlcNAc levels in cells. Further researches revealed that there was a reciprocal relationship between GFPT1 and PD-L1. Mechanistically, we disclosed that GFPT1 enhanced PD-L1 protein stability through O-glycosylation. More interestingly, GFPT1 accelerated BC cell immune escape via upregulation of O-glycosylation-modified PD-L1. In vivo, silencing of GFPT1 attenuated immune escape of BC cells by reducing PD-L1 levels.ConclusionGFPT1 promoted BC progression and immune escape via O-glycosylation-modified PD-L1. GFPT1 may be a potential target for BC therapy.

Abstract P1138: Transcriptomic Insights Into How Previously Increased Cardiac Protein O-GlcNAcylation Enhances Susceptibility To Cardiac Dysfunction In A Response To Pressure-Overload

Increased levels of cardiac protein posttranslational β-linked N-acetylglucosamine (O-GlcNAc) modification are implicated in adverse remodeling seen in hypertrophy and diabetes. Our previous work found elevated O-GlcNAc levels in hearts from diabetic mice, and a chronic increase in O-GlcNAc caused adverse cardiac remodeling. In diabetes, increased cardiac disease risk persists even after patients return to tight glycemic control. Thus, we hypothesize that a transient increase in O-GlcNAc is sufficient to exacerbate adverse cardiac remodeling under subsequent pressure-overload, and we propose that this is due to epigenetic modifications causing persistent changes in gene expression. We used an inducible, cardiomyocyte specific, dominant negative O-GlcNAcase (dnOGA) mouse. After a 2-wk induction (which leads to a 1.8-fold increase in O-GlcNAc levels) and subsequent 2-wk washout, Control (Con) and dnOGA mice undergo transverse-aortic constriction (TAC) or Sham surgery. After 8-wks, cardiac function is measured via echocardiography, gravimetric measures at harvest, and RNA-seq analysis is done on cardiac tissue. In both Con+TAC and dnOGA+TAC vs. Sham groups, systolic function is decreased ( -12%, p < 0.001, -11%, p < 0.01 ), and ventricular weight is increased ( +43%, +69%, both p < 0.0001 ), consistent with cardiac hypertrophy. Interestingly, we see further exacerbation of cardiac hypertrophy ( +29%, p < 0.0001 ) and pulmonary edema ( +36%, p < 0.01 ) between dnOGA+TAC vs. Con+TAC groups. Also, RNA-seq results show distinct transcriptomic profiles between the experimental groups via hierarchical clustering ( |FC| > 1.50, FDR < 0.1 ). A candidate gene approach shows enhanced and exacerbated changes in genes associated with cardiac disease states like heart failure and fibrosis (e.g., Atp2a2, Col1a1, & Nox4 ), consistent with cardiac remodeling. Our results support the hypothesis that transient increase in cardiac O-GlcNAc levels increases susceptibility to subsequent cardiac pathology. Moreover, the exacerbation of pathology is consistent with epigenetic mediated effects of transient O-GlcNAc induction; and pertaining to diabetes, provide a possible mechanism into the continued increased of cardiac disease risk.

Nutrient sensitive protein O-GlcNAcylation modulates the transcriptome through epigenetic mechanisms during embryonic neurogenesis.

Protein O-GlcNAcylation is a dynamic, nutrient-sensitive mono-glycosylation deposited on numerous nucleo-cytoplasmic and mitochondrial proteins, including transcription factors, epigenetic regulators, and histones. However, the role of protein O-GlcNAcylation on epigenome regulation in response to nutrient perturbations during development is not well understood. Herein we recapitulated early human embryonic neurogenesis in cell culture and found that pharmacological up-regulation of O-GlcNAc levels during human embryonic stem cells' neuronal differentiation leads to up-regulation of key neurogenic transcription factor genes. This transcriptional de-repression is associated with reduced H3K27me3 and increased H3K4me3 levels on the promoters of these genes, perturbing promoter bivalency possibly through increased EZH2-Thr311 phosphorylation. Elevated O-GlcNAc levels also lead to increased Pol II-Ser5 phosphorylation and affect H2BS112O-GlcNAc and H2BK120Ub1 on promoters. Using an in vivo rat model of maternal hyperglycemia, we show similarly elevated O-GlcNAc levels and epigenetic dysregulations in the developing embryo brains because of hyperglycemia, whereas pharmacological inhibition of O-GlcNAc transferase (OGT) restored these molecular changes. Together, our results demonstrate O-GlcNAc mediated sensitivity of chromatin to nutrient status, and indicate how metabolic perturbations could affect gene expression during neurodevelopment.

Protein O-GlcNAcylation in the heart.

O-GlcNAcylation is a ubiquitous post-translational modification that is extremely labile and plays a significant role in physiology, including the heart. Sustained activation of cardiac O-GlcNAcylation is frequently associated with alterations in cellular metabolism, leading to detrimental effects on cardiovascular function. This is particularly true during conditions such as diabetes, hypertension, cardiac remodelling, heart failure and arrhythmogenesis. Paradoxically, transient elevation of cardiac protein O-GlcNAcylation can also exert beneficial effects in the heart. There is compelling evidence to suggest that a complex interaction between O-GlcNAcylation and phosphorylation also exists in the heart. Beyond direct functional consequences on cardiomyocytes, O-GlcNAcylation also acts indirectly by altering the function of transcription factors that affect downstream signalling. This review focuses on the potential cardioprotective role of protein O-GlcNAcylation during ischaemia-reperfusion injury, the deleterious consequences of chronically elevated O-GlcNAc levels, the interplay between O-GlcNAcylation and phosphorylation in the cardiomyocytes and the effects of O-GlcNAcylation on other major non-myocyte cell types in the heart.

Aspartate Residues Far from the Active Site Drive O-GlcNAc Transferase Substrate Selection.

O-GlcNAc is an abundant post-translational modification found on nuclear and cytoplasmic proteins in all metazoans. This modification regulates a wide variety of cellular processes, and elevated O-GlcNAc levels have been implicated in cancer progression. A single essential enzyme, O-GlcNAc transferase (OGT), is responsible for all nucleocytoplasmic O-GlcNAcylation. Understanding how this enzyme chooses its substrates is critical for understanding, and potentially manipulating, its functions. Here we use protein microarray technology and proteome-wide glycosylation profiling to show that conserved aspartate residues in the tetratricopeptide repeat (TPR) lumen of OGT drive substrate selection. Changing these residues to alanines alters substrate selectivity and unexpectedly increases rates of protein glycosylation. Our findings support a model where sites of glycosylation for many OGT substrates are determined by TPR domain contacts to substrate side chains five to fifteen residues C-terminal to the glycosite. In addition to guiding design of inhibitors that target OGT's TPR domain, this information will inform efforts to engineer substrates to explore biological functions.

Elevated O-GlcNAcylation enhances pro-inflammatory Th17 function by altering the intracellular lipid microenvironment

Chronic, low-grade inflammation increases the risk for atherosclerosis, cancer, and autoimmunity in diseases such as obesity and diabetes. Levels of CD4+ T helper 17 (Th17) cells, which secrete interleukin 17A (IL-17A), are increased in obesity and contribute to the inflammatory milieu; however, the relationship between signaling events triggered by excess nutrient levels and IL-17A-mediated inflammation is unclear. Here, using cytokine, quantitative real-time PCR, immunoprecipitation, and ChIP assays, along with lipidomics and MS-based approaches, we show that increased levels of the nutrient-responsive, post-translational protein modification, O-GlcNAc, are present in naive CD4+ T cells from a diet-induced obesity murine model and that elevated O-GlcNAc levels increase IL-17A production. We also found that increased binding of the Th17 master transcription factor RAR-related orphan receptor γ t variant (RORγt) at the IL-17 gene promoter and enhancer, as well as significant alterations in the intracellular lipid microenvironment, elevates the production of ligands capable of increasing RORγt transcriptional activity. Importantly, the rate-limiting enzyme of fatty acid biosynthesis, acetyl-CoA carboxylase 1 (ACC1), is O-GlcNAcylated and necessary for production of these RORγt-activating ligands. Our results suggest that increased O-GlcNAcylation of cellular proteins may be a potential link between excess nutrient levels and pathological inflammation.

Elevated O‐GlcNAc Exacerbates Pro‐Inflammatory Cytokine Secretion from CD4+ T cells

Chronic inflammation is a feature of obesity and enhances the risk of atherosclerosis, cancer, diabetes, and autoimmunity. Specifically, pro-inflammatory TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell function. Since production of O-GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a sensor of a cell's nutritional status. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells from mice fed a high fat and cholesterol “Western” diet. Naïve CD4+ T cells from obese mice have elevated O-GlcNAc levels compared to cells from mice fed standard chow. When polarized to a TH17 lineage, cells from obese mice secrete more IL-17A, the eponymous TH17 cytokine. Importantly, when naïve CD4+ T cells from lean and obese mice are polarized to a TH17 lineage in the presence of Thiamet-G (TMG, an inhibitor of the enzyme that removes O-GlcNAc (O-GlcNAcase, OGA)) the cells from lean mice secrete levels of IL-17 comparable to those from obese mice and IL-17 secretion was exacerbated in cells from obese mice. Transcript levels of IL-17 are similarly elevated in both lean and obese mice TH17 cells treated with TMG. RORγt (retinoic acid-related orphan receptor gamma) acts as the TH17 master transcription factor, orchestrating the TH17 differentiation program. Uniquely among the CD4+ effector T cell master transcription factors, RORγt is regulated by fatty acid ligands which promote or repress its activity. Acetyl CoA carboxylase 1 (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt. We discovered that ACC1 is O-GlcNAcylated. Furthermore, lipidomics analysis identified clear differences in the lipid environment of T cells isolated from lean and obese mice −/+ TMG. Thus, abnormally elevated O-GlcNAc levels may favor the generation of ligands that enhance RORγt activity at the IL-17 promoter. More directly implicating O-GlcNAc's regulation of RORγt, chromatin immunoprecipation of RORγt and O-GlcNAc transferase (OGT, the enzyme that modifies proteins with O-GlcNAc) at the IL-17 promoter and an enhancer, CNS2 (conserved noncoding sequence 2), is increased with TMG treatment. Collectively, our data suggest that elevated O-GlcNAc levels increase pro-inflammatory IL-17 secretion from TH17 cells through alteration of the lipidome and direct effects on the IL-17 promoter complex. Further study into the molecular mechanism of how O-GlcNAcylation promotes pro-inflammatory TH17 T cell function will provide insight into how nutritional excess worsens inflammation. Support or Funding Information This research was supported by NIH grant R01DK100595-01, NIH grant R01DK091277-03, COBRE P30GM122731, and a KUMC Institute of Reproductive Health and Regenerative Medicine pilot grant. MM is supported by a KUMC Biomedical Research Training Program grant. Elevated O-GlcNAc increases pro-inflammatory IL-17 cytokine secretion from TH17 cells This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

O-GlcNAc on PKCζ Inhibits the FGF4-PKCζ-MEK-ERK1/2 Pathway via Inhibition of PKCζ Phosphorylation in Mouse Embryonic Stem Cells.

SummaryMouse embryonic stem cells (ESCs) differentiate into multiple cell types during organismal development. Fibroblast growth factor 4 (FGF4) signaling induces differentiation from ESCs via the phosphorylation of downstream molecules such as mitogen-activated protein kinase/extracellular signal-related kinase (MEK) and extracellular signal-related kinase 1/2 (ERK1/2). The FGF4-MEK-ERK1/2 pathway is inhibited to maintain ESCs in the undifferentiated state. However, the inhibitory mechanism of the FGF4-MEK-ERK1/2 pathway in ESCs is uncharacterized. O-linked β-N-acetylglucosaminylation (O-GlcNAcylation) is a post-translational modification characterized by the attachment of a single N-acetylglucosamine (GlcNAc) to the serine and threonine residues of nuclear or cytoplasmic proteins. Here, we showed that the O-GlcNAc on the phosphorylation site of PKCζ inhibits PKCζ phosphorylation (activation) and, consequently, the FGF4-PKCζ-MEK-ERK1/2 pathway in ESCs. Our results demonstrate the mechanism for the maintenance of the undifferentiated state of ESCs via the inhibition of the FGF4-PKCζ-MEK-ERK1/2 pathway by O-GlcNAcylation on PKCζ.

Sustained O-GlcNAcylation reprograms mitochondrial function to regulate energy metabolism

Dysfunctional mitochondria and generation of reactive oxygen species (ROS) promote chronic diseases, which have spurred interest in the molecular mechanisms underlying these conditions. Previously, we have demonstrated that disruption of post-translational modification of proteins with β-linked N-acetylglucosamine (O-GlcNAcylation) via overexpression of the O-GlcNAc-regulating enzymes O-GlcNAc transferase (OGT) or O-GlcNAcase (OGA) impairs mitochondrial function. Here, we report that sustained alterations in O-GlcNAcylation either by pharmacological or genetic manipulation also alter metabolic function. Sustained O-GlcNAc elevation in SH-SY5Y neuroblastoma cells increased OGA expression and reduced cellular respiration and ROS generation. Cells with elevated O-GlcNAc levels had elongated mitochondria and increased mitochondrial membrane potential, and RNA-sequencing analysis indicated transcriptome reprogramming and down-regulation of the NRF2-mediated antioxidant response. Sustained O-GlcNAcylation in mouse brain and liver validated the metabolic phenotypes observed in the cells, and OGT knockdown in the liver elevated ROS levels, impaired respiration, and increased the NRF2 antioxidant response. Moreover, elevated O-GlcNAc levels promoted weight loss and lowered respiration in mice and skewed the mice toward carbohydrate-dependent metabolism as determined by indirect calorimetry. In summary, sustained elevation in O-GlcNAcylation coupled with increased OGA expression reprograms energy metabolism, a finding that has potential implications for the etiology, development, and management of metabolic diseases.

O-GlcNAc Regulates CD4+ T Cell Differentiation

Abstract Chronic inflammation is a feature of obesity and enhances the risk of heart disease, cancer, and diabetes. Specifically, pro-inflammatory TH1 and TH17 CD4+ effector T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with pro-inflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification (PTM) of nuclear and cytoplasmic proteins, promotes pro-inflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from multiple metabolic pathways, the PTM acts as a nutrient sensor. To investigate the role of O-GlcNAc in a setting of metabolic disease, we analyzed O-GlcNAc levels in CD4+ T cells in mice fed a high fat diet. CD4+ T cells from obese mice have elevated O-GlcNAc levels and are primed to secrete more IL-17A, the eponymous TH17 cytokine, and IFNγ, the signature TH1 cytokine. We also cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc. We find a significant increase in protein and transcript levels of IL-17A and IFNγ in TMG treated cells. Acetyl CoA carboxylase (ACC1), the rate limiting enzyme in fatty acid synthesis, is known to enhance generation of TH17 cells by producing ligands that increase the activity of RORγt, the transcription factor defining the TH17 lineage. We find that ACC1 is O-GlcNAcylated in CD4+ T cells. Collectively, our data suggest that elevated O-GlcNAc levels prime TH17 CD4+ T cell differentiation by altering ACC1 activity. Further study into how O-GlcNAcylation promotes pro-inflammatory T cell differentiation will provide insight into how nutritional excess exacerbates inflammation.

Fatty acid synthase inhibits the O-GlcNAcase during oxidative stress

The dynamic post-translational modification O-linked β-N-acetylglucosamine (O-GlcNAc) regulates thousands of nuclear, cytoplasmic, and mitochondrial proteins. Cellular stress, including oxidative stress, results in increased O-GlcNAcylation of numerous proteins, and this increase is thought to promote cell survival. The mechanisms by which the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA), the enzymes that add and remove O-GlcNAc, respectively, are regulated during oxidative stress to alter O-GlcNAcylation are not fully characterized. Here, we demonstrate that oxidative stress leads to elevated O-GlcNAc levels in U2OS cells but has little impact on the activity of OGT. In contrast, the expression and activity of OGA are enhanced. We hypothesized that this seeming paradox could be explained by proteins that bind to and control the local activity or substrate targeting of OGA, thereby resulting in the observed stress-induced elevations of O-GlcNAc. To identify potential protein partners, we utilized BioID proximity biotinylation in combination with stable isotopic labeling of amino acids in cell culture (SILAC). This analysis revealed 90 OGA-interacting partners, many of which exhibited increased binding to OGA upon stress. The associations of OGA with fatty acid synthase (FAS), filamin-A, heat shock cognate 70-kDa protein, and OGT were confirmed by co-immunoprecipitation. The pool of OGA bound to FAS demonstrated a substantial (∼85%) reduction in specific activity, suggesting that FAS inhibits OGA. Consistent with this observation, FAS overexpression augmented stress-induced O-GlcNAcylation. Although the mechanism by which FAS sequesters OGA remains unknown, these data suggest that FAS fine-tunes the cell's response to stress and injury by remodeling cellular O-GlcNAcylation.

Dissecting PUGNAc-mediated inhibition of the pro-survival action of insulin.

Previous studies utilizing PUGNAc, the most widely used β-N-acetylglucosaminidase (OGA) inhibitor to increase global O-N-acetylglucosamine (GlcNAc) levels, have reported a variety of effects including insulin resistance as a direct result of elevated O-GlcNAc levels. The notion of OGA inhibition causing insulin resistance was not replicated in studies in which elevated global O-GlcNAc levels were achieved using two other OGA inhibitors. Related to insulin action, work by others has suggested that O-GlcNAc elevation may inhibit the anti-apoptotic action of insulin. Thus, we examined the pro-survival action of insulin upon serum deprivation in the presence of PUGNAc as well as two selective OGA inhibitors (GlcNAcstatin-g and Thiamet-G), and a selective lysosomal hexosaminidase inhibitor (INJ2). We established that PUGNAc inhibits the pro-survival action of insulin but this effect is not recapitulated by the selective OGA inhibitors suggesting that elevation in O-GlcNAc levels alone is not responsible for PUGNAc's effect on the anti-apoptotic action of insulin. Further, we demonstrate that a selective hexosaminidase A/B (HexA/B) inhibitor does not impact insulin action suggesting that PUGNAc's effect is not due to inhibition of lysosomal hexosaminidase. Finally, we tested a combination of selective OGA and lysosomal hexosaminidase inhibitors but were not able to recapitulate the inhibition of insulin action generated by PUGNAc alone. These results strongly suggest that the defect in insulin action upon PUGNAc treatment does not derive from its inhibition of OGA or HexA/B, and that there is an unknown target of PUGNAc that is the likely culprit in inhibiting the protective effect of insulin from apoptosis.

O-GlcNAc regulates CD4+ T cell differentiation

Abstract Chronic inflammation is a feature of diabetes and obesity and enhances the risk of atherosclerosis and cancer. Specifically, proinflammatory Th1 and Th17 CD4+ T cells are increased in metabolic diseases. However, a clear molecular mechanism linking metabolic changes with proinflammatory T cells is lacking. We hypothesize that elevated levels of O-linked β-N-acetylglucosamine (O-GlcNAc), a post-translational modification of nuclear and cytoplasmic proteins, promotes proinflammatory CD4+ T cell differentiation. Since production of O-GlcNAc involves input from carbohydrate, amino acid, fatty acid, and nucleic acid metabolism, the modification acts as a general sensor of a cell’s nutritional status. To investigate O-GlcNAc’s role, we cultured mouse splenic CD4+ T cells with Thiamet-G (TMG), a selective inhibitor of the enzyme that removes O-GlcNAc (O-GlcNAcase, OGA), before activating with anti-CD3 and anti-CD28 under permissive Th0 conditions. We find a significant two-fold increase in protein and transcript levels of IL-17A, the eponymous Th17 cytokine. Intracellular cytokine staining shows two-fold increases in IFNγ+ cells, IL-17+ cells, and IFNγ+IL-17+ cells. Phosphorylation of STAT3 and STAT4, the transcriptionally active form of STATs which define the Th17 and Th1 lineage programs respectively, is increased in TMG-treated cells within 10 minutes of T cell receptor activation. Collectively, our data suggest that elevated O-GlcNAc levels prime Th17, Th1, and Th1-like Th17 CD4+ T cell differentiation by increasing activated STAT3 and STAT4. Further study into the molecular mechanism of how O-GlcNAcylation promotes proinflammatory T cells may provide insight into how nutritional excess exacerbates inflammation.

O-Linked β-N-acetylglucosamine (O-GlcNAc) Acts as a Glucose Sensor to Epigenetically Regulate the Insulin Gene in Pancreatic Beta Cells

The post-translational protein modification O-linked β-N-acetylglucosamine (O-GlcNAc) is a proposed nutrient sensor that has been shown to regulate multiple biological pathways. This dynamic and inducible enzymatic modification to intracellular proteins utilizes the end product of the nutrient sensing hexosamine biosynthetic pathway, UDP-GlcNAc, as its substrate donor. Type II diabetic patients have elevated O-GlcNAc-modified proteins within pancreatic beta cells due to chronic hyperglycemia-induced glucose overload, but a molecular role for O-GlcNAc within beta cells remains unclear. Using directed pharmacological approaches in the mouse insulinoma-6 (Min6) cell line, we demonstrate that elevating nuclear O-GlcNAc increases intracellular insulin levels and preserves glucose-stimulated insulin secretion during chronic hyperglycemia. The molecular mechanism for these observed changes appears to be, at least in part, due to elevated O-GlcNAc-dependent increases in Ins1 and Ins2 mRNA levels via elevations in histone H3 transcriptional activation marks. Furthermore, RNA deep sequencing reveals that this mechanism of altered gene transcription is restricted and that the majority of genes regulated by elevated O-GlcNAc levels are similarly regulated by a shift from euglycemic to hyperglycemic conditions. These findings implicate the O-GlcNAc modification as a potential mechanism for hyperglycemic-regulated gene expression in the beta cell.

O-GlcNAcylation Negatively Regulates Cardiomyogenic Fate in Adult Mouse Cardiac Mesenchymal Stromal Cells.

In both preclinical and clinical studies, cell transplantation of several cell types is used to promote repair of damaged organs and tissues. Nevertheless, despite the widespread use of such strategies, there remains little understanding of how the efficacy of cell therapy is regulated. We showed previously that augmentation of a unique, metabolically derived stress signal (i.e., O-GlcNAc) improves survival of cardiac mesenchymal stromal cells; however, it is not known whether enhancing O-GlcNAcylation affects lineage commitment or other aspects of cell competency. In this study, we assessed the role of O-GlcNAc in differentiation of cardiac mesenchymal stromal cells. Exposure of these cells to routine differentiation protocols in culture increased markers of the cardiomyogenic lineage such as Nkx2.5 and connexin 40, and augmented the abundance of transcripts associated with endothelial and fibroblast cell fates. Differentiation significantly decreased the abundance of O-GlcNAcylated proteins. To determine if O-GlcNAc is involved in stromal cell differentiation, O-GlcNAcylation was increased pharmacologically during the differentiation protocol. Although elevated O-GlcNAc levels did not significantly affect fibroblast and endothelial marker expression, acquisition of cardiomyocyte markers was limited. In addition, increasing O-GlcNAcylation further elevated smooth muscle actin expression. In addition to lineage commitment, we also evaluated proliferation and migration, and found that increasing O-GlcNAcylation did not significantly affect either; however, we found that O-GlcNAc transferase—the protein responsible for adding O-GlcNAc to proteins—is at least partially required for maintaining cellular proliferative and migratory capacities. We conclude that O-GlcNAcylation contributes significantly to cardiac mesenchymal stromal cell lineage and function. O-GlcNAcylation and pathological conditions that may affect O-GlcNAc levels (such as diabetes) should be considered carefully in the context of cardiac cell therapy.

Elevated O-GlcNAcylation promotes colonic inflammation and tumorigenesis by modulating NF-κB signaling.

O-GlcNAcylation is a reversible post-translational modification. O-GlcNAc addition and removal is catalyzed by O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), respectively. More recent evidence indicates that regulation of O-GlcNAcylation is important for inflammatory diseases and tumorigenesis. In this study, we revealed that O-GlcNAcylation was increased in the colonic tissues of dextran sodium sulfate (DSS)-induced colitis and azoxymethane (AOM)/DSS-induced colitis-associated cancer (CAC) animal models. Moreover, the O-GlcNAcylation level was elevated in human CAC tissues compared with matched normal counterparts. To investigate the functional role of O-GlcNAcylation in colitis, we used OGA heterozygote mice, which have an increased level of O-GlcNAcylation. OGA(+/-) mice have higher susceptibility to DSS-induced colitis than OGA(+/+) mice. OGA(+/-) mice exhibited a higher incidence of colon tumors than OGA(+/+) mice. In molecular studies, elevated O-GlcNAc levels were shown to enhance the activation of NF-κB signaling through increasing the binding of RelA/p65 to its target promoters. We also found that Thr-322 and Thr352 in the p65-O-GlcNAcylation sites are critical for p65 promoter binding. These results suggest that the elevated O-GlcNAcylation level in colonic tissues contributes to the development of colitis and CAC by disrupting regulation of NF-κB-dependent transcriptional activity.

Global O-GlcNAc Levels Modulate Transcription of the Adipocyte Secretome during Chronic Insulin Resistance.

Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O-linked β-N-acetylglucosamine (O-GlcNAc) is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify multiple rodent adipocytokines and secreted proteins whose levels are modulated upon the induction of IR by indirectly and directly modulating O-GlcNAc levels. We have validated the relative levels of several of these factors using immunoblotting. Since adipocytokines levels are regulated primarily at the level of transcription and O-GlcNAc alters the function of many transcription factors, we hypothesized that elevated O-GlcNAc levels on key transcription factors are modulating secreted protein expression. Here, we show that upon the elevation of O-GlcNAc levels and the induction of IR in mature 3T3-F442a adipocytes, the transcript levels of multiple secreted proteins reflect the modulation observed at the protein level. We validate the transcript levels in male mouse models of diabetes. Using inguinal fat pads from the severely IR db/db mouse model and the mildly IR diet-induced mouse model, we have confirmed that the secreted proteins regulated by O-GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR. By comparing the promoters of similarly regulated genes, we determine that Sp1 is a common cis-acting element. Furthermore, we show that the LPL and SPARC promoters are enriched for Sp1 and O-GlcNAc modified proteins during insulin resistance in adipocytes. Thus, the O-GlcNAc modification of proteins bound to promoters, including Sp1, is linked to adipocytokine transcription during insulin resistance.

The role of cardiac O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) transferase (OGT) on endurance capacity and physical activity (LB681)

The cardiovascular benefits obtained from endurance exercise are well established. Physiological cardiac hypertrophy occurs in response to the beneficial stress of exercise at both the cellular and transcriptional levels. The mechanisms triggering cardiac remodeling to induce a physiological or pathological hypertrophic phenotype remain largely unknown. The posttranslational attachment of O‐Linked β‐N‐acetylglucosamine (O‐GlcNAc) to serine or threonine residues of proteins is catalyzed by O‐GlcNAc transferase (OGT). O‐GlcNAcylation is an important mediator of signal transduction and protein‐protein interactions. However, the relevance of elevated O‐GlcNAc levels in the heart is complex as it is implicated in both cardiac dysfunction, and cardioprotection. Preliminary data and recent literature also suggests that inducible cardiomyocyte‐specific knockdown of OGT (OGT‐KD) alters gene transcription, which results in a heart failure phenotype. We investigated the impact of inducible, cardiomyocyte‐specific OGT‐KD on exercise capacity in a mouse model. Ten week old OGT‐KD mice were randomly assigned to a one‐time run to exhaustion treadmill (TM) cohort, or a 4 week voluntary wheel running (WR) cohort, or appropriate control groups. The TM cohort tolerated a decreased workload overall and exhibited increased ventricular heart weight with an increase in the ratio of heart weight (HW) to tibia length (TL). OGT‐KD mice in the WR cohort had decreased body weight, decreased overall activity, and increased HW:TL. These data suggest that inducible cardiac‐specific OGT‐KD in a mouse model significantly decreases endurance capacity and physical activity, while causing hypertrophy of the heart muscle as early as 11 weeks of age.Grant Funding Source: NIH/NHLBI HL104549

O‐GlcNAc levels modulate adipocytokine transcription

Increased flux through the hexosamine biosynthetic pathway and the corresponding increase in intracellular glycosylation of proteins via O‐GlcNAc is sufficient to induce insulin resistance (IR) in multiple systems. Previously, our group used shotgun proteomics to identify multiple rodent adipocytokines whose levels are modulated upon the induction of IR by indirectly and directly modulating O‐GlcNAc levels. We have validated the relative levels of several of these adipocytokines using immunoblotting. Since adipocytokines levels are regulated primarily at the level of transcription and O‐GlcNAc alters the function of many transcription factors, we hypothesized that elevated O‐GlcNAc levels on key transcription factors are modulating adipocytokine expression. Here, we show that upon the elevation of O‐GlcNAc levels and the induction of IR in mature 3T3‐F442a adipocytes, the steady‐state transcript levels of multiple adipocytokines, as measured by quantitative RT‐PCR, reflect the modulation observed at the protein level. We have gone on to validate the adipocytokine transcript levels in mouse models of diabetes. Using inguinal fat pads from the severely IR db/db mouse model and the mildly IR diet‐induced mouse model, we have confirmed that the adipocytokines regulated by O‐GlcNAc modulation in cell culture are likewise modulated in the whole animal upon a shift to IR.R01DK075069(LW)

Elevation of Global O-GlcNAc in Rodents Using a Selective O-GlcNAcase Inhibitor Does Not Cause Insulin Resistance or Perturb Glucohomeostasis

SummaryThe O-GlcNAc modification is proposed to be a nutrient sensor with studies suggesting that global increases in O-GlcNAc levels cause insulin resistance and impaired glucohomeostasis. We address this hypothesis by using a potent and selective inhibitor of O-GlcNAcase, known as NButGT, in a series of in vivo studies. Treatment of rats and mice with NButGT, for various time regimens and doses, dramatically increases O-GlcNAc levels throughout all tissues but does not perturb insulin sensitivity or alter glucohomeostasis. NButGT also does not affect the severity or onset of insulin resistance induced by a high-fat diet. These results suggest that pharmacological increases in global O-GlcNAc levels do not cause insulin resistance nor do they appear to disrupt glucohomeostasis. Therefore, the protective benefits of elevated O-GlcNAc levels may be achieved without deleteriously affecting glucohomeostasis.