Increased Protein O-GlcNAcylation Research Articles

How Nanotechniques Could Vitalize the O-GlcNAcylation-Targeting Approach for Cancer Therapy.

Accumulated data indicated that many types of cancers have increased protein O-GlcNAcylation at cell surface and inside cells. The aberrant O-GlcNAcylation is considered a potential therapeutic target. Although several types of compounds capable of inhibiting O-GlcNAcylation have been developed, their low solubility, poor permeability and delivery efficiency have impeded the application for in vivo and pre-clinical studies. Nanocarriers have the advantages of controllable drug release and active cancer-targeting capability. Moreover, nanoparticles can improve drug delivery efficiency and reduce the non-specific distribution in normal tissues by the enhanced permeability and retention (EPR) effect in cancer. Taking the advantage of O-GlcNAc-specific antibodies or lectins, nanoparticles could further improve their cancer-targeting capability. Although nanocarriers targeting the canonical N- and O-linked glycosylation have been extensively investigated for cancer detection and therapy, application of nanotechniques for the specific targeting of O-GlcNAcylation has not been actively pursued. This review summarizes the general features of GlcNAcylation and its alterations in cancers. Analyses are focused on the following areas: How the nanocarriers may improve the solubility and/or cell permeability of O-GlcNAc transferase (OGT) inhibitors; The modification of nanocarriers with lectins or antibodies for active targeting of O-GlcNAc; The nanocarriers-mediated co-delivery of OGT inhibitors and conventional drugs, which may lead to synergistic effects. Unsolved issues impeding the research progression on O-GlcNAcylation-targeting scheme are also discussed.

Targeting Protein O-GlcNAcylation, a Link between Type 2 Diabetes Mellitus and Inflammatory Disease.

Unresolved hyperglycaemia, a hallmark of type 2 diabetes mellitus (T2DM), is a well characterised manifestation of altered fuel homeostasis and our understanding of its role in the pathologic activation of the inflammatory system continues to grow. Metabolic disorders like T2DM trigger changes in the regulation of key cellular processes such as cell trafficking and proliferation, and manifest as chronic inflammatory disorders with severe long-term consequences. Activation of inflammatory pathways has recently emerged as a critical link between T2DM and inflammation. A substantial body of evidence has suggested that this is due in part to increased flux through the hexosamine biosynthetic pathway (HBP). The HBP, a unique nutrient-sensing metabolic pathway, produces the activated amino sugar UDP-GlcNAc which is a critical substrate for protein O-GlcNAcylation, a dynamic, reversible post-translational glycosylation of serine and threonine residues in target proteins. Protein O-GlcNAcylation impacts a range of cellular processes, including inflammation, metabolism, trafficking, and cytoskeletal organisation. As increased HBP flux culminates in increased protein O-GlcNAcylation, we propose that targeting O-GlcNAcylation may be a viable therapeutic strategy for the prevention and management of glucose-dependent pathologies with inflammatory components.

Diaminocyclopentane-derived O-GlcNAcase inhibitors for combating tau hyperphosphorylation in Alzheimer's disease.

We developed potent and selective aminocyclopentane-derived inhibitors of human O-N-acetyl-β-D-glucosaminidase (OGA) implicated in Alzheimer's disease. For example compound 13 was a nanomolar OGA inhibitor with 92 000-fold selectivity over human HexB. It was non-toxic and increased protein O-GlcNAcylation in the culture of murine neural cells, showing new alternatives in the treatment of tauopathies.

Temporal regulation of protein O-GlcNAc levels during pressure-overload cardiac hypertrophy.

Protein posttranslational modifications (PTMs) by O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) rise during pressure‐overload hypertrophy (POH) to affect hypertrophic growth. The hexosamine biosynthesis pathway (HBP) branches from glycolysis to make the moiety for O‐GlcNAcylation. It is speculated that greater glucose utilization during POH augments HBP flux to increase O‐GlcNAc levels; however, recent results suggest glucose availability does not primarily regulate cardiac O‐GlcNAc levels. We hypothesize that induction of key enzymes augment protein O‐GlcNAc levels primarily during active myocardial hypertrophic growth and remodeling with early pressure overload. We further speculate that downregulation of protein O‐GlcNAcylation inhibits ongoing hypertrophic growth during prolonged pressure overload with established hypertrophy. We used transverse aortic constriction (TAC) to create POH in C57/Bl6 mice. Experimental groups were sham, 1‐week TAC (1wTAC) for early hypertrophy, or 6‐week TAC (6wTAC) for established hypertrophy. We used western blots to determine O‐GlcNAc regulation. To assess the effect of increased protein O‐GlcNAcylation with established hypertrophy, mice received thiamet‐g (TG) starting 4 weeks after TAC. Protein O‐GlcNAc levels were significantly elevated in 1wTAC versus Sham with a fall in 6wTAC. OGA, which removes O‐GlcNAc from proteins, fell in 1wTAC versus sham. GFAT is the rate‐limiting HBP enzyme and the isoform GFAT1 substantially rose in 1wTAC. With established hypertrophy, TG increased protein O‐GlcNAc levels but did not affect cardiac mass. In summary, protein O‐GlcNAc levels vary during POH with elevations occurring during active hypertrophic growth early after TAC. O‐GlcNAc levels appear to be regulated by changes in key enzyme levels. Increasing O‐GlcNAc levels during established hypertrophy did not restart hypertrophic growth.

MO620ANTIFIBROTIC EFFECTS OF SGLT2 INHIBITION IN THE KIDNEY AND PROXIMAL TUBULAR CELLS*

Abstract Background and Aims Diabetic kidney disease (DKD) is a major cause of chronic kidney disease and end stage renal disease, therefore identification of novel therapeutic strategies that reduce the risk of DKD is a research priority. Recent large clinical trials suggest that improved renal outcomes by sodium-glucose cotransporter 2 inhibitors (SGLT2i) are partly beyond their glucose lowering effects. Enhanced glucose reabsorption in diabetes leads to tubular hypoxia triggering fibrotic response. Hyperglycemia is in strong association with increased protein O-GlcNAcylation, a post-translational modification contributing to renal fibrosis. Considering the proximal tubular involvement in DKD pathogenesis and the key role of SGLT2 in glucose metabolism, here we investigated the effects of SGLT2i on tubular hypoxia and O-GlcNAcylation. Method Diabetes (D) was induced by streptozotocin (65 mg/bwkg, ip.) in adult, male Wistar rats. Following the onset of diabetes rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day, po.). Metabolic parameters and renal function were evaluated. Novel urinary biomarkers of extracellular matrix remodeling (Pro-C3, uC3M, tumstatin) and profibrotic growth factors (TGF-β, CTGF, PDGF) were determined. Histological evaluation of glomerular damage (PAS), tubulointerstitial fibrosis (Masson’s trichrome, Picrosirius red) and fibronectin accumulation were performed. The effect of hyperglycemia was tested in human proximal tubular epithelial cells (HK-2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions for 24 hours. HG cells were treated with 10 µM DAPA. O-GlcNAc, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 µM DAPA and were placed in a hypoxic chamber (1% O2) for 2 hours. Hypoxic injury was investigated using three different methods (qRT-PCR, Western blot, immunofluorescence analysis). HIF-1α, EPO, VEGFA and profibrotic factors were measured. Results DAPA decreased blood glucose levels (D: 37±2.7 vs. D+DAPA: 18±5.6 mmol/L; p<0.05) and improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p<0.01). In parallel, novel urinary biomarkers of extracellular matrix remodeling, profibrotic growth factor expressions and extensive fibrotic tissue accumulation were reduced in the kidney. DAPA minimized hyperglycemia-induced total protein O-GlcNAcylation in HK-2 cells. Hypoxia-induced HIF-1α elevation was suspended by DAPA treatment. Moreover, DAPA treatment prevented HIF-1α translocation to the nucleus, thereby confirming abolished HIF-1α activation. EPO, VEGFA and profibrotic factor levels were also increased in hypoxia and DAPA prevented EPO, TGFB and PDGF elevation. Conclusion These data highlight the role of ameliorated O-GlcNAcylation and diminished tubular hypoxia as important benefits of SGLT2i treatment. Our results support the link between glucose toxicity, tubular hypoxia and fibrosis, a vicious trio, which seem to be targeted by SGLT2i. All these mechanisms are important parts in the puzzle of the complex system behind the protective effect of SGLT2i. OTKA-FK124491-K135398, 2017-1.3.1-VKE-2017-00006, 2020-4.1.1.-TKP2020-6183169273, 2020-4.1.1.-TKP2020-6183069269

Cardiomyocyte Oga haploinsufficiency increases O-GlcNAcylation but hastens ventricular dysfunction following myocardial infarction.

RationaleThe beta-O-linkage of N-acetylglucosamine (i.e., O-GlcNAc) to proteins is a pro-adaptive response to cellular insults. To this end, increased protein O-GlcNAcylation improves short-term survival of cardiomyocytes subjected to acute injury. This observation has been repeated by multiple groups and in multiple models; however, whether increased protein O-GlcNAcylation plays a beneficial role in more chronic settings remains an open question.ObjectiveHere, we queried whether increasing levels of cardiac protein O-GlcNAcylation would be beneficial during infarct-induced heart failure.Methods and resultsTo achieve increased protein O-GlcNAcylation, we targeted Oga, the gene responsible for removing O-GlcNAc from proteins. Here, we generated mice with cardiomyocyte-restricted, tamoxifen-inducible haploinsufficient Oga gene. In the absence of infarction, we observed a slight reduction in ejection fraction in Oga deficient mice. Overall, Oga reduction had no major impact on ventricular function. In additional cohorts, mice of both sexes and both genotypes were subjected to infarct-induced heart failure and followed for up to four weeks, during which time cardiac function was assessed via echocardiography. Contrary to our prediction, the Oga deficient mice exhibited exacerbated—not improved—cardiac function at one week following infarction. When the observation was extended to 4 wk post-MI, this acute exacerbation was lost.ConclusionsThe present findings, coupled with our previous work, suggest that altering the ability of cardiomyocytes to either add or remove O-GlcNAc modifications to proteins exacerbates early infarct-induced heart failure. We speculate that more nuanced approaches to regulating O-GlcNAcylation are needed to understand its role—and, in particular, the possibility of cycling, in the pathophysiology of the failing heart.

Involvement of NDPK-B in Glucose Metabolism-Mediated Endothelial Damage via Activation of the Hexosamine Biosynthesis Pathway and Suppression of O-GlcNAcase Activity.

Our previous studies identified that retinal endothelial damage caused by hyperglycemia or nucleoside diphosphate kinase-B (NDPK-B) deficiency is linked to elevation of angiopoietin-2 (Ang-2) and the activation of the hexosamine biosynthesis pathway (HBP). Herein, we investigated how NDPK-B is involved in the HBP in endothelial cells (ECs). The activities of NDPK-B and O-GlcNAcase (OGA) were measured by in vitro assays. Nucleotide metabolism and O-GlcNAcylated proteins were assessed by UPLC-PDA (Ultra-performance liquid chromatography with Photodiode array detection) and immunoblot, respectively. Re-expression of NDPK-B was achieved with recombinant adenoviruses. Our results show that NDPK-B depletion in ECs elevated UDP-GlcNAc levels and reduced NDPK activity, similar to high glucose (HG) treatment. Moreover, the expression and phosphorylation of glutamine:fructose-6-phosphate amidotransferase (GFAT) were induced, whereas OGA activity was suppressed. Furthermore, overall protein O-GlcNAcylation, along with O-GlcNAcylated Ang-2, was increased in NDPK-B depleted ECs. Pharmacological elevation of protein O-GlcNAcylation using Thiamet G (TMG) or OGA siRNA increased Ang-2 levels. However, the nucleoside triphosphate to diphosphate (NTP/NDP) transphosphorylase and histidine kinase activity of NDPK-B were dispensable for protein O-GlcNAcylation. NDPK-B deficiency hence results in the activation of HBP and the suppression of OGA activity, leading to increased protein O-GlcNAcylation and further upregulation of Ang-2. The data indicate a critical role of NDPK-B in endothelial damage via the modulation of the HBP.

Insulin Resistance Does Not Impair Mechanical Overload-Stimulated Glucose Uptake, but Does Alter the Metabolic Fate of Glucose in Mouse Muscle.

Skeletal muscle glucose uptake and glucose metabolism are impaired in insulin resistance. Mechanical overload stimulates glucose uptake into insulin-resistant muscle; yet the mechanisms underlying this beneficial effect remain poorly understood. This study examined whether a differential partitioning of glucose metabolism is part of the mechanosensitive mechanism underlying overload-stimulated glucose uptake in insulin-resistant muscle. Mice were fed a high-fat diet to induce insulin resistance. Plantaris muscle overload was induced by unilateral synergist ablation. After 5 days, muscles were excised for the following measurements: (1) [3H]-2-deoxyglucose uptake; (2) glycogen; 3) [5-3H]-glucose flux through glycolysis; (4) lactate secretion; (5) metabolites; and (6) immunoblots. Overload increased glucose uptake ~80% in both insulin-sensitive and insulin-resistant muscles. Overload increased glycogen content ~20% and this was enhanced to ~40% in the insulin-resistant muscle. Overload did not alter glycolytic flux, but did increase muscle lactate secretion 40–50%. In both insulin-sensitive and insulin-resistant muscles, overload increased 6-phosphogluconate levels ~150% and decreased NADP:NADPH ~60%, indicating pentose phosphate pathway activation. Overload increased protein O-GlcNAcylation ~45% and this was enhanced to ~55% in the insulin-resistant muscle, indicating hexosamine pathway activation. In conclusion, insulin resistance does not impair mechanical overload-stimulated glucose uptake but does alter the metabolic fate of glucose in muscle.

Overexpression of p53 due to excess protein O-GlcNAcylation is associated with coronary microvascular disease in type 2 diabetes.

We previously reported that increased protein O-GlcNAcylation in diabetic mice led to vascular rarefaction in the heart. In this study, we aimed to investigate whether and how coronary endothelial cell (EC) apoptosis is enhanced by protein O-GlcNAcylation and thus induces coronary microvascular disease (CMD) and subsequent cardiac dysfunction in diabetes. We hypothesize that excessive protein O-GlcNAcylation increases p53 that leads to CMD and reduced cardiac contractility. We conducted in vivo functional experiments in control mice, TALLYHO/Jng (TH) mice, a polygenic type 2 diabetic (T2D) model, and EC-specific O-GlcNAcase (OGA, an enzyme that catalyzes the removal of O-GlcNAc from proteins)-overexpressing TH mice, as well as in vitro experiments in isolated ECs from these mice. TH mice exhibited a significant increase in coronary EC apoptosis and reduction of coronary flow velocity reserve (CFVR), an assessment of coronary microvascular function, in comparison to wild-type mice. The decreased CFVR, due at least partially to EC apoptosis, was associated with decreased cardiac contractility in TH mice. Western blot experiments showed that p53 protein level was significantly higher in coronary ECs from TH mice and T2D patients than in control ECs. High glucose treatment also increased p53 protein level in control ECs. Furthermore, overexpression of OGA decreased protein O-GlcNAcylation and down-regulated p53 in coronary ECs, and conferred a protective effect on cardiac function in TH mice. Inhibition of p53 with pifithrin-α attenuated coronary EC apoptosis and restored CFVR and cardiac contractility in TH mice. The data from this study indicate that inhibition of p53 or down-regulation of p53 by OGA overexpression attenuates coronary EC apoptosis and improves CFVR and cardiac function in diabetes. Lowering coronary endothelial p53 levels via OGA overexpression could be a potential therapeutic approach for CMD in diabetes.

593-P: Diabetes Enhances Translation of the mRNA Encoding CD40 in Müller Glia to Promote Retinal Inflammation

This study investigated the hypothesis that diabetes-induced hyperglycemia promotes retinal inflammation by augmenting translation of the immune costimulatory molecule CD40. In response to diabetes, Müller glia initiate retinal inflammation via activation of CD40. CD40 activation in Müller glia leads to ATP release, stimulation of P2X7 purinergic receptors on microglia/macrophages, and their subsequent release of inflammatory cytokines. Increased CD40 expression is a common feature of inflammatory diseases driven by CD40. Indeed, CD40 protein expression is elevated in Müller glia of diabetic mice, yet the mechanisms responsible for this increase have not been fully explored. In microglia/macrophages, inflammatory cytokines enhance CD40 gene transcription via cis-elements in the CD40 promoter. However, cytokine-mediated CD40 transcriptional upregulation is not sufficient to explain a model wherein retinal inflammation is initiated by CD40 expression in Müller glia. A recent genome-wide functional screening revealed that the untranslated region of the mRNA encoding CD40 was capable of promoting translation independent of the mRNA m7GTP cap. Our laboratory has previously demonstrated that diabetes-induced hyperglycemia promotes O-GlcNAcylation of the translational repressor 4E-BP1, leading to enhanced cap-independent mRNA translation. In the present study, in vivo translation of the CD40 mRNA was enhanced in Müller glia of STZ-diabetic RiboTag mice concomitant with increased protein O-GlcNAcylation. Enhanced O-GlcNAcylation was sufficient to promote CD40 mRNA translation in both retina and cells in culture, and O-GlcNAcylation-induced CD40 mRNA translation was ablated by 4E-BP deletion. In the retina of diabetic 4E-BP-deficient mice, CD40 translation and retinal pathology were normalized. Thus, therapeutic approaches targeting diabetes-induced defects in mRNA translation may be beneficial for preventing diabetic retinopathy progression. Disclosure S.K. Dierschke: None. A. Toro: None. W.P. Miller: None. M.D. Dennis: None. Funding American Diabetes Association/Pathway to Stop Diabetes (1-14-INI-04 to M.D.D.)

25-LB: SGLT2 Inhibitors Are Renoprotective via Mitigation of Tubular Hypoxia and Protein O-GlcNAcylation

Tubular hypoxia has a major pathogenic role in diabetic kidney disease (DKD). Hypoxia is in strong association with elevated glucose reabsorption and increased protein O-GlcNAcylation which could contribute to renal fibrosis. We recently showed that SGLT2 inhibitors (SGLT2i) are renoprotective in experimental type 1 diabetes. Considering emerging evidence of proximal tubular involvement in DKD and the major role of SGLT2 in glucose metabolism, here we investigated the direct effects of SGLT2i on hypoxia and O-GlcNAcylation. Diabetes (D) was induced by streptozotocin in adult, male Wistar rats. Rats were treated for six weeks with dapagliflozin (D+DAPA, 1 mg/bwkg/day). Renal function and fibrosis were evaluated. The effect of hyperglycaemia was tested in human proximal tubular epithelial cells (HK-2) kept under normal glucose (5.5 mM), high glucose (35 mM) or high mannitol (osmotic control, 35 mM) conditions. HG cells were treated with 10 µM DAPA. O-GlcNAc, O GlcNAc transferase (OGT) and O GlcNAcase (OGA) were measured. To test the effect of hypoxia cells were treated with 10 µM DAPA and were placed in a hypoxic chamber for 2 hours. HIF1-α, EPO, VEGFA and PAI-1 were measured. Immunocytochemistry (ICC) of HIF1-α was performed. DAPA improved renal function (creatinine clearance: D: 3.8±0.4 vs. D+DAPA: 8.9±1.0 mL/min; p<0.01) and decreased fibrosis. DAPA minimized hyperglycemia-induced total protein O-GlcNAcylation and OGT, while OGA was elevated in HK-2 cells. Hypoxia-induced HIF-1α elevation was suspended by DAPA treatment. Abolishment of HIF-1α upregulation by DAPA was confirmed by ICC staining. EPO, VEGFA and PAI-1 levels were also increased in hypoxia and DAPA prevented EPO and PAI-1 elevation. Here we identified a novel mechanism of SGLT2i by which the direct reduction of tubular hypoxia and inhibition of OGT by DAPA results in decreased O-GlcNAcylation in proximal tubular cells. These processes contribute to improved renal function and alleviated kidney fibrosis. Disclosure D.B. Balogh: None. J. Hodrea: None. L. Lenart: None. A. Hosszu: None. C. Mezei: None. L. Wagner: None. A.J. Szabo: None. A. Fekete: None. Funding Hungarian Academy of Sciences (LP008/2017, OTKA-K112629-FK124491-NN-114607, VKE-2017-00006); Semmelweis University; New National Excellence Program of the Hungarian Ministry of Human Capacities (?NKP-18-3)

Effect of Chronic Hyperglycemia on Glucose Metabolism in Subjects With Normal Glucose Tolerance.

Chronic hyperglycemia causes insulin resistance, but the inheritability of glucotoxicity and the underlying mechanisms are unclear. We examined the effect of 3 days of hyperglycemia on glucose disposal, enzyme activities, insulin signaling, and protein O-GlcNAcylation in skeletal muscle of individuals without (FH−) or with (FH+) family history of type 2 diabetes. Twenty-five subjects with normal glucose tolerance received a [3-3H]glucose euglycemic insulin clamp, indirect calorimetry, and vastus-lateralis biopsies before and after 3 days of saline (n = 5) or glucose (n = 10 FH− and 10 FH+) infusion to raise plasma glucose by ∼45 mg/dL. At baseline, FH+ had lower insulin-stimulated glucose oxidation and total glucose disposal (TGD) but similar nonoxidative glucose disposal and basal endogenous glucose production (bEGP) compared with FH−. After 3 days of glucose infusion, bEGP and glucose oxidation were markedly increased, whereas nonoxidative glucose disposal and TGD were lower versus baseline, with no differences between FH− and FH+ subjects. Hyperglycemia doubled skeletal muscle glycogen content and impaired activation of glycogen synthase (GS), pyruvate dehydrogenase, and Akt, but protein O-GlcNAcylation was unchanged. Insulin resistance develops to a similar extent in FH− and FH+ subjects after chronic hyperglycemia, without increased protein O-GlcNAcylation. Decreased nonoxidative glucose disposal due to impaired GS activation appears to be the primary deficit in skeletal muscle glucotoxicity.

Abstract 274: Regulation of Myocardial Ketone Body Oxidation Capacity by Increased Protein O-GlcNAcylation in Diabetic Heart

Diabetes increases the risk of heart failure independently of underlying coronary artery disease. Perturbations in myocardial substrate utilization have been proposed to contribute to the pathogenesis of cardiac dysfunction. However, the exact mechanisms linking impaired substrate utilization to disease progression remain elusive. Compared to the well-established roles of glucose and fatty acids metabolism in cardiac physiology and pathology, the importance of ketone body (KB) metabolism is less well understood. Several findings suggest the importance of KB in the cardiac health and disease. More recently, two independent studies have simultaneously demonstrated increased reliance on KB utilization by the failing heart in the absence of diabetes. Despite these observations, our understanding of the importance of KB metabolism in a diabetic heart is incomplete. Interestingly, our findings from the hearts of streptozotocin-induced diabetic mice confirmed elevated cardiac β-hydroxybutyrate (a major KB) levels and transcriptional suppression of Oxct1 gene expression that encodes for SCOT (succinyl-CoA:3-oxoacid CoA transferase; a rate limiting enzyme of KB oxidation), consistent with a mismatch between KB availability and utilization. To determine a specific role for glucose delivery in this regulation we used an inducible transgenic mouse model with cardiac-restricted expression of the glucose transporter 4 (GLUT4; mG4H). Interestingly, mG4H mice had reduced cardiac SCOT protein levels and oxidation of KB in isolated working heart suggesting that glucose is sufficient to regulate the ketone metabolic pathway. Hyperglycemia associated with diabetes alter protein function via post-translational O -GlcNAcylation. Interestingly, we found that myocardial SCOT expression was also reduced in a third mouse model with increased cardiac O -GlcNAc levels (overexpression of a dominant negative form of O -GlcNAcase, which normally removes O -GlcNAc; dnOGAh) suggesting that increased protein O -GlcNAcylation is directly involved. Collectively, these studies establish a novel role of glucose and its downstream regulator, O -GlcNAc, in attenuation of KB metabolism in the diabetic heart.

High-Fat Diet/Palmitate–Induced ER Stress Promotes Protein O-GlcNAcylation in Retina and Retinal Muller Cells

The incidence of type 2 diabetes, the most common cause of diabetic retinopathy (DR), is rapidly on the rise due to the overconsumption of calorie rich diets. Using an animal model of diet-induced obesity/pre-type 2 diabetes, we evaluated the mechanism responsible for increased O-GlcNAcylation of retinal proteins following consumption of a diet high in saturated fat (HFD), as dysregulated O-GlcNAcylation contributes to the development of DR. In the retina of mice fed a HFD for 4 weeks, increased O-GlcNAcylation was observed concomitant with markers of endoplasmic reticulum (ER) stress. Similarly, in TR-MUL retinal cells in culture, addition of the saturated fatty acid palmitate or the ceramide analog Cer6 to culture medium increased protein O-GlcNAcylation and ER stress. One potential mechanism whereby ER stress increases O-GlcNAcylation is by upregulating flux through the hexosamine biosynthetic pathway (HBP) via increased expression of the rate-limiting enzyme glutamine-fructose-6-phosphate amidotransferase (GFAT). In the retina of mice fed a HFD and in TR-MUL cells exposed to Cer6, mRNA and protein expression of GFAT2, but not GFAT1 were increased. Similarly, in TR-MUL cells treated with the ER stress inducer thapsigargin (TG) increased O-GlcNAcylation was observed concomitant with an increase in GFAT2. Alternatively, ER stress inhibitor prevented the effect. GFAT2 knockdown via siRNA attenuated O-GlcNAcylation induced by either TG or Cer6. The glial cell specific transcription factor OASIS (old astrocyte specifically-induced substance) plays a central role in coordinating the unfolded protein response. Unlike GFAT1, GFAT2 contains a putative binding site for OASIS. Both HFD and Cer6 increased OASIS expression, and OASIS was sufficient to promote GFAT2 expression and increase O-GlcNAcylation. Overall, the results support a model whereby HFD-induced ER stress acts to promote O-GlcNAcylation in retina through an OASIS/GFAT2-dependent mechanism. Disclosure W. Dai: None. A. Toro: None. S.K. Dierschke: None. M.D. Dennis: None.

AMPK activation counteracts cardiac hypertrophy by reducing O-GlcNAcylation

AMP-activated protein kinase (AMPK) has been shown to inhibit cardiac hypertrophy. Here, we show that submaximal AMPK activation blocks cardiomyocyte hypertrophy without affecting downstream targets previously suggested to be involved, such as p70 ribosomal S6 protein kinase, calcineurin/nuclear factor of activated T cells (NFAT) and extracellular signal-regulated kinases. Instead, cardiomyocyte hypertrophy is accompanied by increased protein O-GlcNAcylation, which is reversed by AMPK activation. Decreasing O-GlcNAcylation by inhibitors of the glutamine:fructose-6-phosphate aminotransferase (GFAT), blocks cardiomyocyte hypertrophy, mimicking AMPK activation. Conversely, O-GlcNAcylation-inducing agents counteract the anti-hypertrophic effect of AMPK. In vivo, AMPK activation prevents myocardial hypertrophy and the concomitant rise of O-GlcNAcylation in wild-type but not in AMPKα2-deficient mice. Treatment of wild-type mice with O-GlcNAcylation-inducing agents reverses AMPK action. Finally, we demonstrate that AMPK inhibits O-GlcNAcylation by mainly controlling GFAT phosphorylation, thereby reducing O-GlcNAcylation of proteins such as troponin T. We conclude that AMPK activation prevents cardiac hypertrophy predominantly by inhibiting O-GlcNAcylation.

Higher O-GlcNAc Levels Are Associated with Defects in Progenitor Proliferation and Premature Neuronal Differentiation during in-Vitro Human Embryonic Cortical Neurogenesis.

The nutrient responsive O-GlcNAcylation is a dynamic post-translational protein modification found on several nucleocytoplasmic proteins. Previous studies have suggested that hyperglycemia induces the levels of total O-GlcNAcylation inside the cells. Hyperglycemia mediated increase in protein O-GlcNAcylation has been shown to be responsible for various pathologies including insulin resistance and Alzheimer's disease. Since maternal hyperglycemia during pregnancy is associated with adverse neurodevelopmental outcomes in the offspring, it is intriguing to identify the effect of increased protein O-GlcNAcylation on embryonic neurogenesis. Herein using human embryonic stem cells (hESCs) as model, we show that increased levels of total O-GlcNAc is associated with decreased neural progenitor proliferation and premature differentiation of cortical neurons, reduced AKT phosphorylation, increased apoptosis and defects in the expression of various regulators of embryonic corticogenesis. As defects in proliferation and differentiation during neurodevelopment are common features of various neurodevelopmental disorders, increased O-GlcNAcylation could be one mechanism responsible for defective neurodevelopmental outcomes in metabolically compromised pregnancies such as diabetes.

O-GlcNAc Regulation of Autophagy and α-synuclein Homeostasis; Implications for Parkinson’s Disease

Post-translational modification on protein Ser/Thr residues by O-linked attachment of s-N-acetyl-glucosamine (O-GlcNAcylation) is a key mechanism integrating redox signaling, metabolism and stress responses. One of the most common neurodegenerative diseases that exhibit aberrant redox signaling, metabolism and stress response is Parkinson’s disease, suggesting a potential role for O-GlcNAcylation in its pathology. To determine whether abnormal O-GlcNAcylation occurs in Parkinson’s disease, we analyzed lysates from the postmortem temporal cortex of Parkinson’s disease patients and compared them to age matched controls and found increased protein O-GlcNAcylation levels. To determine whether increased O-GlcNAcylation affects neuronal function and survival, we exposed rat primary cortical neurons to thiamet G, a highly selective inhibitor of the enzyme which removes the O-GlcNAc modification from target proteins, O-GlcNAcase (OGA). We found that inhibition of OGA by thiamet G at nanomolar concentrations significantly increased protein O-GlcNAcylation, activated MTOR, decreased autophagic flux, and increased α-synuclein accumulation, while sparing proteasomal activities. Inhibition of MTOR by rapamycin decreased basal levels of protein O-GlcNAcylation, decreased AKT activation and partially reversed the effect of thiamet G on α-synuclein monomer accumulation. Taken together we have provided evidence that excessive O-GlcNAcylation is detrimental to neurons by inhibition of autophagy and by increasing α-synuclein accumulation.

O-GlcNAc regulation of autophagy and \u03b1-synuclein homeostasis; implications for Parkinson\u2019s disease

Post-translational modification on protein Ser/Thr residues by O-linked attachment of ß-N-acetyl-glucosamine (O-GlcNAcylation) is a key mechanism integrating redox signaling, metabolism and stress responses. One of the most common neurodegenerative diseases that exhibit aberrant redox signaling, metabolism and stress response is Parkinson’s disease, suggesting a potential role for O-GlcNAcylation in its pathology. To determine whether abnormal O-GlcNAcylation occurs in Parkinson’s disease, we analyzed lysates from the postmortem temporal cortex of Parkinson’s disease patients and compared them to age matched controls and found increased protein O-GlcNAcylation levels. To determine whether increased O-GlcNAcylation affects neuronal function and survival, we exposed rat primary cortical neurons to thiamet G, a highly selective inhibitor of the enzyme which removes the O-GlcNAc modification from target proteins, O-GlcNAcase (OGA). We found that inhibition of OGA by thiamet G at nanomolar concentrations significantly increased protein O-GlcNAcylation, activated MTOR, decreased autophagic flux, and increased α-synuclein accumulation, while sparing proteasomal activities. Inhibition of MTOR by rapamycin decreased basal levels of protein O-GlcNAcylation, decreased AKT activation and partially reversed the effect of thiamet G on α-synuclein monomer accumulation. Taken together we have provided evidence that excessive O-GlcNAcylation is detrimental to neurons by inhibition of autophagy and by increasing α-synuclein accumulation.

O-GlcNAcylation and cardiovascular disease.

The post-translational modification of serine and threonine residues of proteins found in numerous subcellular locations by O-linked N-acetylglucosamine (O-GlcNAc) is emerging as a key mediator of many cardiovascular pathophysiological processes. Early studies implicated increased protein O-GlcNAcylation as contributing to the cardiovascular complications associated with diabetes, whereas subsequent studies demonstrated that acute increases in O-GlcNAc levels were protective against ischemia/reperfusion injury. There is now a growing understanding that O-GlcNAc modification of proteins influences numerous cellular functions, including transcription, protein turnover, calcium handling, and bioenergetics. As a result, a more nuanced view of the role of protein O-GlcNAcylation in the cardiovascular system is emerging along with the recognition that it is required for normal cellular function and homeostasis. Consequently, the impact of changes in O-GlcNAc cycling due to stress or disease on the heart is complex and highly dependent on the specific context of these events. The goal of this review is to provide an overview of some of the more recent advances in our understanding of the role O-GlcNAcylation plays in mediating cardiovascular function and disease.

405 - Hyper-O-GlcNAcylation Attenuates Autophagic Flux in an MTOR Dependent Manner and Leads to Accumulation of α-Synuclein in Neurons

O-GlcNAcylation is one of the unique forms of glycosylation which involves the β-O-linkage of N-acetylglucosamine (O-GlcNAc) to cellular proteins. This unique post translational modification is involved in a vast array of physiological and pathological functions. To determine the impact of increased O-GlcNAcylation on neuronal function and survival, we cultured rat primary cortical neurons and exposed them with thiamet G, a highly selective OGA inhibitor. We found that thiamet G significantly increased protein O-GlcNAcylation, increased p-mTOR/mTOR, and decreased autophagic flux. This was accompanied by an accumulation of aggregation-prone, Parkinson’s disease causing α-synuclein protein. To determine whether enhancing autophagy in the presence of hyper-O-GlcNAcylation may attenuate α-synuclein accumulation, we exposed neurons to rapamycin, an mTOR dependent autophagy inducer. Rapamycin alone significantly decreased basal O-GlcNAcylated protein levels, without decreasing thiamet-G induced hyper-O-GlcNAcylation. Interestingly, rapamycin partially rescued hyper-O-GlcNAcylation-induced autophagic inhibition as indicated by a partial decrease of p62 levels in the presence of rapamycin in neurons exposed to thiamet G. Furthermore, thiamet-G induced α-synuclein accumulation was also partially decreased by rapamycin. Taken together the results of this study suggest that mTOR activation may be one of the mechanisms involved in hyper-O-GlcNAcylation induced autophagic inhibition and α-synuclein accumulation.