Activation Of Hexosamine Biosynthetic Pathway Research Articles

The dual role of the hexosamine biosynthetic pathway in cardiac physiology and pathophysiology

The heart is a highly metabolic organ with extensive energy demands and hence relies on numerous fuel substrates including fatty acids and glucose. However, oxidative stress is a natural by-product of metabolism that, in excess, can contribute towards DNA damage and poly-ADP-ribose polymerase activation. This activation inhibits key glycolytic enzymes, subsequently shunting glycolytic intermediates into non-oxidative glucose pathways such as the hexosamine biosynthetic pathway (HBP). In this review we provide evidence supporting the dual role of the HBP, i.e. playing a unique role in cardiac physiology and pathophysiology where acute upregulation confers cardioprotection while chronic activation contributes to the onset and progression of cardio-metabolic diseases such as diabetes, hypertrophy, ischemic heart disease, and heart failure. Thus although the HBP has emerged as a novel therapeutic target for such conditions, proposed interventions need to be applied in a context- and pathology-specific manner to avoid any potential drawbacks of relatively low cardiac HBP activity.

Hexosamine pathway activation improves memory but does not extend lifespan in mice.

Glucosamine feeding and genetic activation of the hexosamine biosynthetic pathway (HBP) have been linked to improved protein quality control and lifespan extension. However, as an energy sensor, the HBP has been implicated in tumor progression and diabetes. Given these opposing outcomes, it is imperative to explore the long‐term effects of chronic HBP activation in mammals. Thus, we asked if HBP activation affects metabolism, coordination, memory, and survival in mice. N‐acetyl‐D‐glucosamine (GlcNAc) supplementation in the drinking water had no adverse effect on weight in males but increased weight in young females. Glucose or insulin tolerance was not affected up to 20 months of age. Of note, we observed improved memory in young male mice supplemented with GlcNAc. Survival was not changed by GlcNAc treatment. To assess the effects of genetic HBP activation, we overexpressed the pathway's key enzyme GFAT1 and a constitutively activated mutant form in all mouse tissues. We detected elevated levels of the HBP product UDP‐GlcNAc in mouse brains, but did not find any effects on behavior, memory, or survival. Together, while dietary GlcNAc supplementation did not extend survival in mice, it positively affected memory and is generally well tolerated.

Effect of estradiol and IGF1 on glycogen synthesis in bovine uterine epithelial cells.

Glucose is an important nutrient for the endometrium and embryo during pregnancy. This study shows that estradiol (E2)/IGF1 signaling stimulates glycogen synthesis in the uterine epithelium of cows, which could provide glucose when needed. Glycogen storage in the uterine epithelium peaks near estrus and is a potential source of glucose for the endometrium and embryos. However, the hormonal regulation of glycogen synthesis in the uterine epithelium is poorly understood. Our objective was to evaluate the effect of E2 and insulin-like growth factor 1 (IGF1) on glycogenesis in immortalized bovine uterine epithelial (BUTE) cells. Treatment of BUTE cells with E2 (0.1-10 nM) did not increase glycogen levels. However, treatment of BUTE cells with IGF1 (50 or 100 ng/mL) resulted in a >2-fold increase in glycogen. To determine if the uterine stroma produced IGF1 in response to E2, bovine uterine fibroblasts were treated with E2, which increased IGF1 levels. Immunohistochemistry showed higher levels of IGF1 in the stroma on day 1 than on day 11, which coincides with higher glycogen levels in the uterine epithelium. Western blots revealed that IGF1 treatment increased the levels of phospho-AKT, phospho-GSKβ, hexokinase 1, and glycogen synthase in BUTE cells. Metabolomic (GC-MS) analysis showed that IGF1 increased 3-phosphoglycerate and lactate, potentially indicative of increased flux through glycolysis. We also found higher levels of N-acetyl-glucosamine and protein glycosylation after IGF1 treatment, indicating increased hexosamine biosynthetic pathway activity. In conclusion, IGF1 is produced by uterine fibroblasts due to E2, and IGF1 increases glucose metabolism and glycogenesis in uterine epithelial cells. Glycogen stored in the uterine epithelium due to E2/IGF1 signaling at estrus could provide glucose to the endometrium or be secreted into the uterine lumen as a component of histotroph.

GFPT2/GFAT2 and AMDHD2 act in tandem to control the hexosamine pathway.

The hexosamine biosynthetic pathway (HBP) produces the essential metabolite UDP-GlcNAc and plays a key role in metabolism, health, and aging. The HBP is controlled by its rate-limiting enzyme glutamine fructose-6-phosphate amidotransferase (GFPT/GFAT) that is directly inhibited by UDP-GlcNAc in a feedback loop. HBP regulation by GFPT is well studied but other HBP regulators have remained obscure. Elevated UDP-GlcNAc levels counteract the glycosylation toxin tunicamycin (TM), and thus we screened for TM resistance in haploid mouse embryonic stem cells (mESCs) using random chemical mutagenesis to determine alternative HBP regulation. We identified the N-acetylglucosamine deacetylase AMDHD2 that catalyzes a reverse reaction in the HBP and its loss strongly elevated UDP-GlcNAc. To better understand AMDHD2, we solved the crystal structure and found that loss-of-function (LOF) is caused by protein destabilization or interference with its catalytic activity. Finally, we show that mESCs express AMDHD2 together with GFPT2 instead of the more common paralog GFPT1. Compared with GFPT1, GFPT2 had a much lower sensitivity to UDP-GlcNAc inhibition, explaining how AMDHD2 LOF resulted in HBP activation. This HBP configuration in which AMDHD2 serves to balance GFPT2 activity was also observed in other mESCs and, consistently, the GFPT2:GFPT1 ratio decreased with differentiation of human embryonic stem cells. Taken together, our data reveal a critical function of AMDHD2 in limiting UDP-GlcNAc production in cells that use GFPT2 for metabolite entry into the HBP.

Slt2 Is Required to Activate ER-Stress-Protective Mechanisms through TORC1 Inhibition and Hexosamine Pathway Activation.

Slt2, the MAPK of the cell wall integrity (CWI) pathway, connects different signaling pathways and performs different functions in the protective response of S. cerevisiae to stress. Previous work has evidenced the relation of the CWI pathway and the unfolded protein response (UPR), a transcriptional program activated upon endoplasmic reticulum (ER) stress. However, the mechanisms of crosstalk between these pathways and the targets regulated by Slt2 under ER stress remain unclear. Here, we demonstrated that ectopic expression of GFA1, the gene encoding the first enzyme in the synthesis of UDP-GlcNAc by the hexosamine biosynthetic pathway (HBP) or supplementation of the growth medium with glucosamine (GlcN), increases the tolerance of slt2 mutant cells to different ER-stress inducers. Remarkably, GlcN also alleviates the sensitivity phenotype of cells lacking IRE1 or HAC1, the main actors in controlling the UPR. The exogenous addition of GlcN reduced the abundance of glycosylated proteins and triggered autophagy. We also found that TORC1, the central stress and growth controller, is inhibited by tunicamycin exposure in cells of the wild-type strain but not in those lacking Slt2. Consistent with this, the tunicamycin-induced activation of autophagy and the increased synthesis of ATP in response to ER stress were absent by knock-out of SLT2. Altogether, our data placed Slt2 as an essential actor of the ER stress response by regulating the HBP activity and the TORC1-dependent signaling.

MiR-27b-3p Improved High Glucose-Induced Spermatogenic Cell Damage via Regulating Gfpt1/HBP Signaling

Introduction: Diabetes mellitus (DM)-induced testicular damage is characterized by abnormal apoptosis of spermatogenic cells. Here, we clarified the roles and the molecular mechanism of microRNA (miR)-27b-3p in high glucose (HG)-induced spermatogenic cell damage. Methods: GC-1 spg cells were treated with 30 mmol/L glucose for 24 h. Cell viability was assessed by 2.3 3-(4, 5-dimethylthiazolyl2)-2, 5-diphenyltetrazolium bromide (MTT) assay. And, levels of O-linked N-acetylglucosamine (OGT), apoptosis-related proteins, and autophagy-related proteins were evaluated using Western blot. Levels of tumor necrosis factor-α (TNF-α), IL-1β, IL-6, and UDP-N-acetylglucosamine (UDP-GlcNAc) were assessed by enzyme linked immunosorbent (ELISA) assay. Levels of reactive oxygen species (ROS), malonic dialdehyde (MDA) and activity of superoxide dismutase (SOD) in cells were determined using kits. Cell apoptosis was determined using flow cytometry assay. Besides, dual luciferase reporter assay was employed to verify the binding relationship between miR-27b-3p and glutamine-fructose-6-phosphate transaminase 1 (Gfpt1). Results: miR-27b-3p was markedly downregulated in HG-treated GC-1 spg cells. HG treatment caused decreased cell viability, increased oxidative stress and inflammation, and induced autophagy and apoptosis, which were abolished by miR-27b-3p overexpression. miR-27b-3p suppressed the activation of hexosamine biosynthetic pathway (HBP) signaling in HG-treated spermatogenic cells. miR-27b-3p directly bound to Gfpt1 and negatively regulated its expression. Conclusion: miR-27b-3p could improve HG-induced spermatogenic cell damage via regulating Gfpt1/HBP signaling, providing a new treatment strategy for the treatment of DM-induced testicular damage.

Laminar Flow on Endothelial Cells Suppresses eNOS O-GlcNAcylation to Promote eNOS Activity.

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In vivo deep network tracing reveals phosphofructokinase-mediated coordination of biosynthetic pathway activity in the myocardium

Glucose metabolism comprises numerous amphibolic metabolites that provide precursors for not only the synthesis of cellular building blocks but also for ATP production. In this study, we tested how phosphofructokinase-1 (PFK1) activity controls the fate of glucose-derived carbon in murine hearts in vivo. PFK1 activity was regulated by cardiac-specific overexpression of kinase- or phosphatase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase transgenes in mice (termed GlycoLo or GlycoHi mice, respectively). Dietary delivery of 13C6-glucose to these mice, followed by deep network metabolic tracing, revealed that low rates of PFK1 activity promote selective routing of glucose-derived carbon to the purine synthesis pathway to form 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR). Consistent with a mechanism of physical channeling, we found multimeric protein complexes that contained phosphoribosylaminoimidazole carboxylase (PAICS)—an enzyme important for AICAR biosynthesis, as well as chaperone proteins such as Hsp90 and other metabolic enzymes. We also observed that PFK1 influenced glucose-derived carbon deposition in glycogen, but did not affect hexosamine biosynthetic pathway activity. These studies demonstrate the utility of deep network tracing to identify metabolic channeling and changes in biosynthetic pathway activity in the heart in vivo and present new potential mechanisms by which metabolic branchpoint reactions modulate biosynthetic pathways.

Hexosamine Biosynthetic Pathway-Derived O-GlcNAcylation Is Critical for RANKL-Mediated Osteoclast Differentiation.

O-linked-N-acetylglucosaminylation (O-GlcNAcylation) performed by O-GlcNAc transferase (OGT) is a nutrient-responsive post-translational modification (PTM) via the hexosamine biosynthetic pathway (HBP). Various transcription factors (TFs) are O-GlcNAcylated, affecting their activities and significantly contributing to cellular processes ranging from survival to cellular differentiation. Given the pleiotropic functions of O-GlcNAc modification, it has been studied in various fields; however, the role of O-GlcNAcylation during osteoclast differentiation remains to be explored. Kinetic transcriptome analysis during receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL)-mediated osteoclast differentiation revealed that the nexus of major nutrient metabolism, HBP was critical for this process. We observed that the critical genes related to HBP activation, including Nagk, Gfpt1, and Ogt, were upregulated, while the global O-GlcNAcylation was increased concomitantly during osteoclast differentiation. The O-GlcNAcylation inhibition by the small-molecule inhibitor OSMI-1 reduced osteoclast differentiation in vitro and in vivo by disrupting the translocation of NF-κB p65 and nuclear factor of activated T cells c1 (NFATc1) into the nucleus by controlling their PTM O-GlcNAcylation. Furthermore, OSMI-1 had a synergistic effect with bone target therapy on osteoclastogenesis. Lastly, knocking down Ogt with shRNA (shOgt) mimicked OSMI-1’s effect on osteoclastogenesis. Targeting O-GlcNAcylation during osteoclast differentiation may be a valuable therapeutic approach for osteoclast-activated bone diseases.

Crosstalk of the IκB Kinase with Spliced X-Box Binding Protein 1 Couples Inflammation with Glucose Metabolic Reprogramming in Epithelial-Mesenchymal Transition.

Epithelial-mesenchymal transition (EMT) plays a critical role in airway injury, repair, and structural remodeling. IκB kinase (IKK)-NFκB signaling regulates late EMT-associated gene expression. However, IKK-mediated mesenchymal transition occurs earlier than NFκB/RelA subunit-dependent EMT gene expression, leading us to investigate the hypothesis that IKK plays an independent mechanism in transforming growth factor-β (TGFβ)-induced EMT. Time-resolved dissection of early proteome and phosphoproteome changes in response to TGFβ and a specific IKK inhibitor, BMS-345541, revealed that IKK regulates cascades of 23 signaling pathways essential in EMT, including TGFβ signaling, p38 mitogen associate protein kinase (MAPK), Toll receptor signaling, and integrin pathways. We identified early IKK-dependent phosphorylation of core regulatory proteins in essential EMT signaling cassettes, including ATF2, JUN, NFKB1/p105, and others. Interestingly, we found that IKKβ directly complexes with and phosphorylates the spliced X-box-binding protein 1 (XBP1s). XBP1s is an arm of the unfolded protein response (UPR) that activates the hexosamine biosynthetic pathway (HBP), a pathway that mediates protein N-glycosylation and survival from ER stress-induced apoptosis in EMT. We found that inhibition of IKK activity abolishes the phosphorylation of XBP1-T48, blocks XBP1s nuclear translocation, and inhibits the activation of HBP. Our study elucidates a previously unrecognized IKKβ-XBP1s-HBP crosstalk pathway that couples inflammation and glucose metabolic reprogramming in ETM. Because XBP1-HBP controls N-glycosylation of the extracellular matrix (ECM) in EMT, this novel IKKβ-XBP1-HBP pathway may contain therapeutic targets whose inhibition could prevent ECM remodeling in lung fibrosis or other airway remodeling diseases.

Fumarate hydratase-deficient renal cell carcinoma cells respond to asparagine by activation of the unfolded protein response and stimulation of the hexosamine biosynthetic pathway

BackgroundThe loss-of-function mutation of fumarate hydratase (FH) is a driver of hereditary leiomyomatosis and renal cell carcinoma (HLRCC). Fumarate accumulation results in activation of stress-related mechanisms leading to upregulation of cell survival-related genes. To better understand how cells compensate for the loss of FH in HLRCC, we determined the amino acid nutrient requirements of the FH-deficient UOK262 cell line (UOK262) and its FH-repleted control (UOK262WT).MethodsWe determined growth rates and survival of cell lines in response to amino acid depletion and supplementation. RNAseq was used to determine the transcription changes contingent on Asn and Gln supplementation, which was further followed with stable isotope resolved metabolomics (SIRM) using both [U- 13C,15N] Gln and Asn.ResultsWe found that Asn increased the growth rate of both cell lines in vitro. Gln, but not Asn, increased oxygen consumption rates and glycolytic reserve of both cell lines. Although Asn was taken up by the cells, there was little evidence of Asn-derived label in cellular metabolites, indicating that Asn was not catabolized. However, Asn strongly stimulated Gln labeling of uracil and precursors, uridine phosphates and hexosamine metabolites in the UOK262 cells and to a much lesser extent in the UOK262WT cells, indicating an activation of the hexosamine biosynthetic pathway (HBP) by Asn. Asn in combination with Gln, but not Asn or Gln alone, stimulated expression of genes associated with the endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) in UOK262 to a greater extent than in FH-restored cells. The changes in expression of these genes were confirmed by RT-PCR, and the stimulation of the UPR was confirmed orthogonally by demonstration of an increase in spliced XBP1 (sXBP1) in UOK262 cells under these conditions. Asn exposure also increased both the RNA and protein expression of the HBP regulator GFPT2, which is a transcriptional target of sXBP1.ConclusionsAsn in the presence of Gln induces an ER stress response in FH-deficient UOK262 cells and stimulates increased synthesis of UDP-acetyl glycans indicative of HBP activity. These data demonstrate a novel effect of asparagine on cellular metabolism in FH-deficient cells that could be exploited therapeutically.

O-GlcNAc elevation through activation of the hexosamine biosynthetic pathway enhances cancer cell chemoresistance

Chemoresistance has become a major obstacle to the success of cancer therapy, but the mechanisms underlying chemoresistance are not yet fully understood. O-GlcNAcylation is a post-translational modification that is regulated by the hexosamine biosynthetic pathway (HBP) and has an important role in a wide range of cellular functions. Here we assessed the role of O-GlcNAcylation in chemoresistance and investigated the underlying cellular mechanisms. The results showed that the HBP has an important role in cancer cell chemoresistance by regulating O-GlcNAcylation. An increase in the levels of O-GlcNAcylation indicates an increased resistance of cancer cells to chemotherapy. Acute treatment with doxorubicin (DOX) or camptothecin (CPT) induced O-GlcNAcylation through HBP activation. In fact, the chemotherapy agents activated the AKT/X-box-binding protein 1 (XBP1) axis and then induced the HBP. Furthermore, the observed elevation of cellular O-GlcNAcylation led to activation of survival signalling pathways and chemoresistance in cancer cells. Finally, suppression of O-GlcNAcylation reduced the resistance of both established and primary cancer cells to chemotherapy. These results provide significant novel insights regarding the important role of the HBP and O-GlcNAcylation in regulating cancer chemoresistance. Thus, O-GlcNAc inhibition might offer a new strategy for improving the efficacy of chemotherapy.

Formal modeling and analysis of the hexosamine biosynthetic pathway: role of O-linked N-acetylglucosamine transferase in oncogenesis and cancer progression

The alteration of glucose metabolism, through increased uptake of glucose and glutamine addiction, is essential to cancer cell growth and invasion. Increased flux of glucose through the Hexosamine Biosynthetic Pathway (HBP) drives increased cellular O-GlcNAcylation (hyper-O-GlcNAcylation) and contributes to cancer progression by regulating key oncogenes. However, the association between hyper-O-GlcNAcylation and activation of these oncogenes remains poorly characterized. Here, we implement a qualitative modeling framework to analyze the role of the Biological Regulatory Network in HBP activation and its potential effects on key oncogenes. Experimental observations are encoded in a temporal language format and model checking is applied to infer the model parameters and qualitative model construction. Using this model, we discover step-wise genetic alterations that promote cancer development and invasion due to an increase in glycolytic flux, and reveal critical trajectories involved in cancer progression. We compute delay constraints to reveal important associations between the production and degradation rates of proteins. O-linked N-acetylglucosamine transferase (OGT), an enzyme used for addition of O-GlcNAc during O-GlcNAcylation, is identified as a key regulator to promote oncogenesis in a feedback mechanism through the stabilization of c-Myc. Silencing of the OGT and c-Myc loop decreases glycolytic flux and leads to programmed cell death. Results of network analyses also identify a significant cycle that highlights the role of p53-Mdm2 circuit oscillations in cancer recovery and homeostasis. Together, our findings suggest that the OGT and c-Myc feedback loop is critical in tumor progression, and targeting these mediators may provide a mechanism-based therapeutic approach to regulate hyper-O-GlcNAcylation in human cancer.

Hyperinsulinemic Normoglycemia Does Not Meaningfully Improve Myocardial Performance During Cardiac Surgery

Glucose-insulin-potassium (GIK) administration during cardiac surgery inconsistently improves myocardial function, perhaps because hyperglycemia negates the beneficial effects of GIK. The hyperinsulinemic normoglycemic clamp (HNC) technique may better enhance the myocardial benefits of GIK. The authors extended previous GIK investigations by (1) targeting normoglycemia while administering a GIK infusion (HNC); (2) using improved echocardiographic measures of myocardial deformation, specifically myocardial longitudinal strain and strain rate; and (3) assessing the activation of glucose metabolic pathways. A total of 100 patients having aortic valve replacement for aortic stenosis were randomly assigned to HNC (high-dose insulin with concomitant glucose infusion titrated to normoglycemia) versus standard therapy (insulin treatment if glucose >150 mg/dl). The primary outcomes were left ventricular longitudinal strain and strain rate, assessed using speckle-tracking echocardiography. Right atrial tissue was analyzed for activation of glycolysis/pyruvate oxidation and alternative metabolic pathways. Time-weighted mean glucose concentrations were lower with HNC (127 ± 19 mg/dl) than standard care (177 ± 41 mg/dl; P < 0.001). Echocardiographic data were adequate in 72 patients for strain analysis and 67 patients for strain rate analysis. HNC did not improve myocardial strain, with an HNC minus standard therapy difference of -1.2% (97.5% CI, -2.9 to 0.5%; P = 0.11). Strain rate was significantly better, but by a clinically unimportant amount: -0.16 s (-0.30 to -0.03 s; P = 0.007). There was no evidence of increased glycolytic, pyruvate oxidation, or hexosamine biosynthetic pathway activation in right atrial samples (HNC, n = 20; standard therapy, 22). Administration of glucose and insulin while targeting normoglycemia during aortic valve replacement did not meaningfully improve myocardial function.

Epithelial Mesenchymal Transition Induces Aberrant Glycosylation through Hexosamine Biosynthetic Pathway Activation

Deregulated cellular metabolism is a hallmark of tumors. Cancer cells increase glucose and glutamine flux to provide energy needs and macromolecular synthesis demands. Several studies have been focused on the importance of glycolysis and pentose phosphate pathway. However, a neglected but very important branch of glucose metabolism is the hexosamine biosynthesis pathway (HBP). The HBP is a branch of the glucose metabolic pathway that consumes ∼2-5% of the total glucose, generating UDP-GlcNAc as the end product. UDP-GlcNAc is the donor substrate used in multiple glycosylation reactions. Thus, HBP links the altered metabolism with aberrant glycosylation providing a mechanism for cancer cells to sense and respond to microenvironment changes. Here, we investigate the changes of glucose metabolism during epithelial mesenchymal transition (EMT) and the role of O-GlcNAcylation in this process. We show that A549 cells increase glucose uptake during EMT, but instead of increasing the glycolysis and pentose phosphate pathway, the glucose is shunted through the HBP. The activation of HBP induces an aberrant cell surface glycosylation and O-GlcNAcylation. The cell surface glycans display an increase of sialylation α2-6, poly-LacNAc, and fucosylation, all known epitopes found in different tumor models. In addition, modulation of O-GlcNAc levels was demonstrated to be important during the EMT process. Taken together, our results indicate that EMT is an applicable model to study metabolic and glycophenotype changes during carcinogenesis, suggesting that cell glycosylation senses metabolic changes and modulates cell plasticity.

0307 : Acute activation of the hexosamine biosynthetic pathway promotes electrical instability

The hexosamine biosynthetic pathway (HBP) is heightened in cardiomyopathies. We aimed at understanding how over-activation of HBP impacts on cardiac electrophysiology, and acts as a trigger or a substrate of cardiac arrhythmias. Overactivation of the HBP by injection of glucosamine (GlcN 270mg/kg) was assessed under basal conditions and during beta-adrenergic stress on the myocardial energetic status (phosphocreatine to ATP ratio) of rats by 31 P nuclear magnetic resonance spectroscopy. Rat hearts were perfused ex vivo to evaluate the effects of the HBP activation (physiologically: glutamine 0.5mM – gln; overactivated: GlcN 1mM) on: cardiac function, MVO 2 , and arrhythmia score (AS) under basal (spontaneous arrhythmias: sAS) and pacing (pAS) conditions. At the cellular level (HL1 cells), HBP overactivation (GlcN) was assessed on conduction velocity characterized by micro-electrode arrays recordings, and properties of voltage gated ionic channels involved in cellular excitability and conduction by patch clamp experiments. In vivo GlcN did not modify PCr/ATP of the left ventricle under basal conditions. This ratio was significantly decreased during beta-adrenergic stress, while a similar increased heart rate was reported. When hearts were isolated and perfused ex vivo , GlcN induced a decrease of the heart rate and MVO 2 temporarily, and an increase of the sAS and pAS. Physiological activation of HBP with gln did not impact the ex vivo cardiac function and the sAS, but further increased the probability of atrial arrhythmias during pacing. In HL1 cells, GlcN reduced the rate of spontaneous depolarization, modified the direction and the velocity of the impulse propagation, and altered activation and inactivation properties of the sodium channel. Acute HBP activation is associated with an increased vulnerability for arrhythmias and alteration of impulse conduction, suggesting its potential role in the atrial and ventricular arrhythmias. The author hereby declares no conflict of interest

Hyperinsulinemic Normoglycemia Does Not Meaningfully Improve Myocardial Performance during Cardiac Surgery: A Randomized Trial.

Glucose-insulin-potassium (GIK) administration during cardiac surgery inconsistently improves myocardial function, perhaps because hyperglycemia negates the beneficial effects of GIK. The hyperinsulinemic normoglycemic clamp (HNC) technique may better enhance the myocardial benefits of GIK. The authors extended previous GIK investigations by (1) targeting normoglycemia while administering a GIK infusion (HNC); (2) using improved echocardiographic measures of myocardial deformation, specifically myocardial longitudinal strain and strain rate; and (3) assessing the activation of glucose metabolic pathways. A total of 100 patients having aortic valve replacement for aortic stenosis were randomly assigned to HNC (high-dose insulin with concomitant glucose infusion titrated to normoglycemia) versus standard therapy (insulin treatment if glucose >150 mg/dl). The primary outcomes were left ventricular longitudinal strain and strain rate, assessed using speckle-tracking echocardiography. Right atrial tissue was analyzed for activation of glycolysis/pyruvate oxidation and alternative metabolic pathways. Time-weighted mean glucose concentrations were lower with HNC (127 ± 19 mg/dl) than standard care (177 ± 41 mg/dl; P < 0.001). Echocardiographic data were adequate in 72 patients for strain analysis and 67 patients for strain rate analysis. HNC did not improve myocardial strain, with an HNC minus standard therapy difference of -1.2% (97.5% CI, -2.9 to 0.5%; P = 0.11). Strain rate was significantly better, but by a clinically unimportant amount: -0.16 s (-0.30 to -0.03 s; P = 0.007). There was no evidence of increased glycolytic, pyruvate oxidation, or hexosamine biosynthetic pathway activation in right atrial samples (HNC, n = 20; standard therapy, 22). Administration of glucose and insulin while targeting normoglycemia during aortic valve replacement did not meaningfully improve myocardial function.

Circulation: Clinical Summaries.

Circulation: Clinical Summaries.

Dynamic interactions of TATA‐Box binding protein with promoters is regulated by GlcNAcylation

O‐GlcNAcylation is a dynamic posttranslational modification tightly correlated to the concentration of UDP‐GlcNAc, the end product of the hexosamine biosynthetic pathway (HBP), which varies according nutritional factors. Through HBP activity and production of UDP‐GlcNAc, OGT senses nutrient availability and forward the signal to downstream effectors by transferring a GlcNAc moiety to the targeted proteins. In return, the O‐GlcNAcase resets the signal. Interestingly, literature points out a regulation of the basal transcription machinery by O‐GlcNAc. TATA‐box binding protein (TBP) is a key player in regulating transcription since it is required for the activity of all three RNA polymerases. DNA‐bound TBP acts as a dock for recruitment of basal transcription factors and the RNA polymerase II to formation of a functional pre‐initiation complex (PIC). DNA‐bound TBP can also be recognized by NC2 and BTAF1. NC2 stabilizes the chromatin interaction of TBP, whereas the association of TBP with BTAF1 promotes its release. Here, we showed that TBP is O‐GlcNAcylated on its N‐terminus domain at T114, T126 and S158. O‐GlcNAcylated TBP is bound to DNA and the sugar inhibits the interaction with BTAF1. TBP site‐specific O‐GlcNAc mutants have been used to point out the T114 as the main regulator of its interaction. Using STZ‐treated rat models, we showed that TBP/BTAF1 association is decreased in diabetes. Owing to the fact that BTAF1 dynamically regulates the chromatin interaction of TBP, we investigated the dynamic behavior of TBP and TBP O‐GlcNAc by Strip‐FRAP and ChIP assays. Collectively, our results indicate that O‐GlcNAcylation of TBP at T114 regulates TBP/BTAF1 dynamics on gene promoters.

In vivo and in vitro evidence that chronic activation of the hexosamine biosynthetic pathway interferes with leptin-dependent STAT3 phosphorylation.

We previously reported that a 2-day peripheral infusion of glucosamine caused leptin resistance in rats, suggesting a role for the hexosamine biosynthetic pathway (HBP) in the development of leptin resistance. Here we tested leptin responsiveness in mice in which HBP activity was stimulated by offering 30% sucrose solution in addition to chow and water or by infusing glucosamine. Mice were leptin resistant after 33 days of access to sucrose. Resistance was associated with increased activity of the HBP and with phosphorylation of transcription factor signal transducer and activator of transcription-3 Tyr705 [pSTAT3(Y705)] but inhibition of suppressor of cytokine signaling 3 in the liver and hypothalamus. Intravenous infusion of glucosamine for 3 h stimulated pSTAT3(Y705) but prevented leptin-induced phosphorylation of STAT3(S727). In an in vitro system, glucose, glucosamine, and leptin each dose dependently increased O-linked β-N-acetylglucosamine (O-GlcNAc) protein and pSTAT3(Y705) in HepG2 cells. To test the effect of glucose on leptin responsiveness cells were incubated in 5.5 mM (LG) or 20 mM (HG) glucose for 18 h and were treated with 0 or 50 ng/ml leptin for 15 min. HG alone and LG + leptin produced similar increases in O-GlcNAc protein, glutamine fructose-6-phosphate amidotransferase (GFAT), and pSTAT3(Y705) compared with LG media. Leptin did not stimulate these proteins in HG cells, suggesting leptin resistance. Leptin-induced pSTAT3(S727) was prevented by HG media. Inhibition of GFAT with azaserine prevented LG + leptin and HG stimulation of pSTAT3. These data demonstrate development of leptin resistance in sucrose-drinking mice and provide new evidence of leptin-induced stimulation of the HBP.