Glutamine:fructose-6-phosphate Amidotransferase Research Articles

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.

Hexosamine biosynthetic pathway activity in leptin resistant sucrose-drinking rats

Rats offered 30% sucrose solution in addition to chow and water become leptin resistant therefore we investigated the effect of sucrose solution consumption on leptin signaling. In Experiment 1 rats were resistant to 3rd ventricle injections of1.5μg leptin after 36days of sucrose and western blot indicated that resistance was associated with increased basal levels of signal transducer and activator of transcription 3 phosphorylation (pSTAT3). In Experiment 2 rats were resistant to a peripheral injection of 2mg leptin/kg after 26days of sucrose. Immunohistochemistry indicated that increased basal pSTAT3 was limited to the medial and lateral arcuate nucleus of the hypothalamus. Increased availability of glucose and fructose can stimulate the hexosamine biosynthetic pathway (HBP) which O-GlcNAc-modifies proteins. This has the potential to change protein bioactivity. We tested whether this pathway could account for the leptin resistance. There was no increase in the expression of HBP enzymes in tissues from sucrose rats in Experiment 1, however, direct activation of the HBP with a 3h intravenous infusion of 30μmol/kg/min glucosamine significantly increased hypothalamic pSTAT3. Although sucrose consumption and activation of the HBP both increase hypothalamic pSTAT3 experiments described here did not provide evidence of a direct link between sucrose consumption, HBP activity and leptin resistance. Unexpectedly, we found that the HBP enzyme glutamine fructose-6-phosphate amidotransferase (GFAT) in liver and O-GlcNAcase in hypothalamus were increased 30min after leptin injection in leptin responsive animals, implying a complex interaction between activity of the HBP and leptin responsiveness.

The hexosamine biosynthetic pathway induces gene promoter activity of acetyl-CoA carboxylase beta

The cardiac isoform of acetyl-CoA carboxylase (ACCβ) produces malonyl-CoA, a potent inhibitor of mitochondrial fatty acid (FA) uptake. Higher ACCβ activity decreases FA utilization, potentially leading to intracellular myocardial lipid accumulation and insulin resistance (IR). Since increased hexosamine biosynthetic pathway (HBP) flux is linked to IR onset, we hypothesized that HBP activation leads to the induction of ACCβ gene promoter activity. Rat H9c2 cardio-myoblasts were transiently transfected with a 1317bp human ACCβ promoter-luciferase construct (pPIIβ−1317)±an expression construct encoding the HBP rate-limiting step, i.e., glutamine:fructose 6-phosphate amidotransferase (GFAT)±various HBP modulators. The administration of l-glutamine (HBP substrate) dose-dependently increased, while HBP inhibitors attenuated pPIIβ−1317 activity. Co-transfections with dominant-negative GFAT constructs diminished pPIIβ−1317 activity. To explore underlying transcriptional mechanisms, we co-transfected with upstream stimulatory factor (USF) expression constructs and found that USF2 induced pPIIβ−1317 activity vs. controls. Moreover, co-transfection of a GFAT expression construct+USF reporter-promoter construct (with consensus USF binding elements) led to induction of pPIIβ−1317 activity vs. controls. We next performed transfections with GFAT±full length ACCβ and seven truncated promoter-luciferase constructs, respectively. Here GFAT-mediated ACCβ promoter induction was blunted when co-transfected with the pPIIβ−38/+65 deletion construct indicating that USF2 binds to the proximal ACCβ promoter region (near start codon). Our study demonstrates that HBP activation induces ACCβ gene promoter activity in H9c2 cells via USF2. We propose that such ACCβ induction may elicit serious downstream effects, e.g. the inhibition of FA β-oxidation and the onset of IR.

Abstract 670: A Metabolic Approach to Examining the Hexosamine Biosynthetic Pathway and Its Role in the Development of Diabetes-Associated Atherosclerosis

Introduction: Diabetes is a disease affecting millions of people worldwide, and is a major independent risk factor for cardiovascular disease (CVD). Despite a vast amount of research, the molecular mechanisms that link diabetes to CVD are not well understood. Current evidence suggests that increased flux through the hexosamine biosynthetic pathway (HBP) contributes to the development of hyperglycemia-associated diabetic complications. Our data suggest that increased HBP flux can induce vascular ER stress and accelerate atherogenesis in a mouse model. We hypothesized that this process can be attenuated by inhibiting the first and rate-limiting enzyme in the HBP - glutamine fructose-6-phosphate amidotransferase (GFAT) - using small molecules. Methods and Results: Using high-performance liquid chromatography coupled to mass spectrometry (HPLC-MS) we have developed a methodology to monitor and quantify the levels of the end product of the HBP, uridine diphosphate N -acetylglucosamine (UDP-GlcNAc). Treatment of HepG2 cells with glucosamine (0.2-5 mM), or adenovirus-directed overexpression of GFAT caused a 3-7 fold increase in UDP-GlcNAc ( P< 0.001 & P< 0.05, respectively). Inhibition of GFAT with three novel compounds - amrinone, lapachol or alloxan - decreased levels of UDP-GlcNAc by 1.5 ( P< 0.05), 3 ( P< 0.05) and 3.5-fold ( P< 0.001), respectively. Furthermore, we show that by modulating HBP flux, we can regulate ER stress levels in cultured HepG2 cells. The physiological relevance of this mechanism is supported by evidence of HBP augmentation in a hyperglycemic mouse model. Conclusions: These results support a role for the HBP in the development of atherosclerosis. Currently, MALDI imaging mass spectrometry is being performed on tissue sections to compare the levels of UDP-GlcNAc directly in hyperglycemic vs. normoglycemic mice. These studies may lead to the identification and validation of novel targets for the development of new pharmaceuticals to prevent diabetic atherosclerosis.

Abstract 251: Investigating the Role of the Hexosamine Biosynthesis Pathway in Diabetic Atherosclerosis

Background: Diabetes mellitus dramatically increases the risk for atherosclerotic cardiovascular disease. We have established that chronic hyperglycemia promotes an increase in glucose flux through the hexosamine biosynthesis pathway (HBP). Central to this pathway is glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme controlling the conversion of glucose to glucosamine. We have shown that glucosamine is a potent inducer of endoplasmic reticulum (ER) stress, which is characterized by the accumulation of misfolded proteins in the ER. Chronic ER stress can initiate a multifaceted, cell-specific, response that results in lipid accumulation, inflammation and apoptosis: the hallmark features of atherosclerosis. We hypothesize that conditions of chronic hyperglycemia, associated with diabetes mellitus, can accelerate the development of atherosclerosis by a mechanism that involves increased HBP flux resulting in glucosamine-induced ER stress and the subsequent activation of pro-atherogenic pathways. Results: STZ-induced hyperglycemic and glucosamine supplemented apoE-/- mice had significantly larger and more advanced atherosclerotic lesions compared to the control mice. Indications of ER stress were increased in atherosclerotic lesions of hyperglycemic and glucosamine-supplemented mice but not in the controls. In glucosamine-supplemented mice the accelerated atherosclerosis was independent of detectable changes in blood glucose concentration, glucose tolerance, plasma insulin, or plasma lipid levels. Treatment with either valproate or 4-phenylbutyrate, two small molecules that have been shown to block ER stress signaling, attenuated hyperglycemic and glucosamine-induced atherogenesis. Our data suggest that elevated concentrations of glucose/glucosamine induce ER stress by interfering with lipid-linked oligosaccharide biosynthesis and N-linked glycosylation. Conclusions: Similar to hyperglycemia, glucosamine-supplementation promotes ER stress and accelerated atherosclerosis in apoE-/- mice. These findings support a model by which conditions of hyperglycemia promote vascular complications via a glucosamine-intermediate.

In vivo and in vitro evidence that leptin stimulates the hexosamine biosynthetic pathway (640.9)

We previously reported that glucosamine administration, which activates the hexosamine biosynthetic pathway (HBP), causes leptin resistance in rats. Here we tested whether nutrient activation of the HBP results in leptin resistance. Male Sprague‐Dawley rats received intraperitoneal infusions of saline or leptin (40 μg/day) for 12 days and were fed chow or a choice diet of chow, 30% sucrose solution, and lard. The choice diet caused leptin resistance and increased hepatic glutamine fructose 6‐phosphate amidotransferase (GFAT), a rate limiting enzyme in the HBP, and O‐linked N‐acetylglucosamine (O‐GlcNAc) protein modification. Leptin infusion increased GFAT and O‐GlcNAcylation in chow‐fed rats, whereas it had no additive effect in choice rats. To test for direct effects of leptin and glucose, HepG2 cells were incubated with high concentrations of glucose (18.5 mM) or with 5.5 mM glucose and leptin (0, 5, 10, 50, 100 or 500 ng/mL). Both leptin and glucose stimulated cellular GFAT and protein O‐GlcNAcylation. Thus, leptin stimulates the HBP both in vivo and in vitro, and activation of the HBP due to increased nutrient flux is associated with leptin resistance.Grant Funding Source: Supported by NIH grant DK 53903

Differential hexosamine biosynthetic pathway gene expression with type 2 diabetes

The hexosamine biosynthetic pathway (HBP) culminates in the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) onto serine/threonine residues of target proteins. The HBP is regulated by several modulators, i.e. O-linked β-N-acetylglucosaminyl transferase (OGT) and β-N-acetylglucosaminidase (OGA) catalyze the addition and removal of O-GlcNAc moieties, respectively; while flux is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFPT), transcribed by two genes, GFPT1 and GFPT2. Since increased HBP flux is glucose-responsive and linked to insulin resistance/type 2 diabetes onset, we hypothesized that diabetic individuals exhibit differential expression of HBP regulatory genes. Volunteers (n=60; n=20 Mixed Ancestry, n=40 Caucasian) were recruited from Stellenbosch and Paarl (Western Cape, South Africa) and classified as control, pre- or diabetic according to fasting plasma glucose and HbA1c levels, respectively. RNA was purified from leukocytes isolated from collected blood samples and OGT, OGA, GFPT1 and GFPT2 expressions determined by quantitative real-time PCR. The data reveal lower OGA expression in diabetic individuals (P<0.01), while pre- and diabetic subjects displayed attenuated OGT expression vs. controls (P<0.01 and P<0.001, respectively). Moreover, GFPT2 expression decreased in pre- and diabetic Caucasians vs. controls (P<0.05 and P<0.01, respectively). We also found ethnic differences, i.e. Mixed Ancestry individuals exhibited a 2.4-fold increase in GFPT2 expression vs. Caucasians, despite diagnosis (P<0.01). Gene expression of HBP regulators differs between diabetic and non-diabetic individuals, together with distinct ethnic-specific gene profiles. Thus differential HBP gene regulation may offer diagnostic utility and provide candidate susceptibility genes for different ethnic groupings.

TRIB3 Mediates Glucose-Induced Insulin Resistance via a Mechanism That Requires the Hexosamine Biosynthetic Pathway

In the current study, we investigated the role of tribbles homolog 3 (TRIB3) in glucose-induced insulin resistance and whether the induction of TRIB3 by glucose is dependent on the nutrient-sensing hexosamine biosynthetic pathway (HBP) known to mediate glucose toxicity in diabetes. In diabetic rats, TRIB3 expression in skeletal muscle was increased after 10 days of hyperglycemia, and glycemia and muscle TRIB3 were both restored toward normal by insulin therapy. In L6 myocytes, the induction of TRIB3 by high glucose or glucosamine was reversible upon removal of these substrates. To assess the role of HBP in the induction of TRIB3, we demonstrated that the ability of high glucose to augment TRIB3 expression was prevented by azaserine, an inhibitor of glutamine: fructose-6-phosphate amidotransferase (GFAT), which is the rate-limiting enzyme in the HBP pathway. TRIB3 expression was also substantially stimulated by glucosamine, which bypasses GFAT, accompanied by a decrease in the insulin-stimulated glucose transport rate, and neither response was affected by azaserine. Further, knockdown of TRIB3 inhibited, and TRIB3 overexpression enhanced, the ability of both high glucose and glucosamine to induce insulin resistance. These data provide the mechanistic link between the HBP flux and insulin resistance and point to TRIB3 as a novel target for treatment of glucose-induced insulin resistance.

Glutamine: fructose-6-phosphate amidotransferase (GFAT): homology modelling and designing of new inhibitors using pharmacophore and docking based hierarchical virtual screening protocol

Glutamine: fructose-6-phosphate amidotransferase (GFAT), also termed GFPT1 and GFAT1, catalyzes the first committed step of the hexosamine biosynthesis pathway in mammals and consequently plays an important role in type 2 diabetes. In the present study, a combination of pharmacophore modelling, homology modelling, and molecular docking analysis was performed to design new glutamine competitive inhibitors of human GFAT, and to investigate important interaction details of inhibitor molecules. A pharmacophore model of GFAT inhibitors was developed, subsequently validated, and utilized for the screening by the PHASE database to identify new molecules. Afterwards, homology modelling was performed to construct the glutamine-binding site of the GFAT protein. The modelled active site was utilized to dock the studied molecules to investigate important receptor-ligand interactions and to scrutinize database-screened molecules on the basis of essential interactions. This systematic in silico protocol helped us to identify new molecules that would be explored for the treatment of type 2 diabetes and its complications.

Novel anti‐atherogenic effects of trivalent chromium in vascular smooth muscle cells: therapeutic implication for diabetic vascular complications

Trivalent chromium (Cr) is proposed to have a therapeutic potential in glycemic and cardiovascular health. While emerging evidence support a protective role of Cr in atherosclerosis, the underlying mechanisms of Cr action in diabetic vascular disease remain unknown. Diabetic patients have increased incidence of vascular smooth muscle cell (VSMC) migration and proliferation, key events leading to initiation and development of atherosclerosis. The present study provides the first evidence that Cr inhibits high glucose (30mM) induced migration and proliferation of human aortic SMC (HASMC) coupled to attenuation of ERK1/2 phosphorylation. We showed earlier that Cr downregulates a potent proatherogenic matricellular protein Thrombospondin‐1 (TSP‐1), known to accelerate initiation of atherosclerosis, concomitant to a decrease in protein O‐glycosylation. We now further demonstrate that this decrease in TSP‐1 expression is accompanied by reduction in expression of Glutamine: fructose‐6‐phosphate amidotransferase (GFAT), the key rate limiting enzyme of hexosamine pathway of glucose metabolism. Additionally, we found that Cr inhibits high glucose‐induced oxidative stress via reduction in cellular reactive oxygen species (ROS). Together, these data suggest TSP‐1 as a potential therapeutic target of Cr and provide a novel anti‐atherogenic mechanism for Cr in diabetic atherosclerotic complications.

Impact of O-GlcNAc on cardioprotection by remote ischaemic preconditioning in non-diabetic and diabetic patients

Post-translational modification of proteins by O-linked β-N-acetylglucosamine (O-GlcNAc) is cardioprotective but its role in cardioprotection by remote ischaemic preconditioning (rIPC) and the reduced efficacy of rIPC in type 2 diabetes mellitus is unknown. In this study we achieved mechanistic insight into the remote stimulus mediating and the target organ response eliciting the cardioprotective effect by rIPC in non-diabetic and diabetic myocardium and the influence of O-GlcNAcylation. The cardioprotective capacity and the influence on myocardial O-GlcNAc levels of plasma dialysate from eight healthy volunteers and eight type 2 diabetic patients drawn before and after subjection to an rIPC stimulus were tested on human isolated atrial trabeculae subjected to ischaemia/reperfusion injury. Dialysate from healthy volunteers exposed to rIPC improved post-ischaemic haemodynamic recovery (40 ± 6 vs. 16 ± 2%; P < 0.01) and increased myocardial O-GlcNAc levels. Similar observations were made with dialysate from diabetic patients before exposure to rIPC (43 ± 3 vs. 16 ± 2%; P < 0.001) but no additional cardioprotection or further increase in O-GlcNAc levels was achieved by perfusion with dialysate after exposure to rIPC (44 ± 4 and 42 ± 5 vs. 43 ± 3%; P = 0.7). The glutamine:fructose-6-phosphate amidotransferase (GFAT) inhibitor azaserine abolished the cardioprotective effects and the increment in myocardial O-GlcNAc levels afforded by plasma from diabetic patients and healthy volunteers treated with rIPC. rIPC and diabetes mellitus per se influence myocardial O-GlcNAc levels through circulating humoral factors. O-GlcNAc signalling participates in mediating rIPC-induced cardioprotection and maintaining a state of inherent chronic activation of cardioprotection in diabetic myocardium, restricting it from further protection by rIPC.

Excess protein O-GlcNAcylation and the progression of diabetic cardiomyopathy

We examined the role that enzymatic protein O-GlcNAcylation plays in the development of diabetic cardiomyopathy in a mouse model of Type 2 diabetes mellitus (DM2). Mice injected with low-dose streptozotocin and fed a high-fat diet developed mild hyperglycemia and obesity consistent with DM2. Studies were performed from 1 to 6 mo after initiating the DM2 protocol. After 1 mo, DM2 mice showed increased body weight, impaired fasting blood glucose, and hyperinsulinemia. Echocardiographic evaluation revealed left ventricular diastolic dysfunction by 2 mo and O-GlcNAcylation of several cardiac proteins and of nuclear transcription factor Sp1. By 4 mo, systolic dysfunction was observed and sarcoplasmic reticulum Ca(2+) ATPase expression decreased by 50%. Fibrosis was not observed at any timepoint in DM2 mice. Levels of the rate-limiting enzyme of the hexosamine biosynthetic pathway, glutamine:fructose-6-phosphate amidotransferase (GFAT) were increased as early as 2 mo. Fatty acids, which are elevated in DM2 mice, can possibly be linked to excessive protein O-GlcNAcylation levels, as cultured cardiac myocytes in normal glucose treated with oleic acid showed increased O-GlcNAcylation and GFAT levels. These data indicate that the early onset of diastolic dysfunction followed by the loss of systolic function, in the absence of cardiac hypertrophy or fibrosis, is associated with increased cardiac protein O-GlcNAcylation and increased O-GlcNAcylation levels of key calcium-handling proteins. A link between excessive protein O-GlcNAcylation and cardiac dysfunction is further supported by results showing that reducing O-GlcNAcylation by O-GlcNAcase overexpression improved cardiac function in the diabetic mouse. In addition, fatty acids play a role in stimulating excess O-GlcNAcylation. The nature and time course of changes observed in cardiac function suggest that protein O-GlcNAcylation plays a mechanistic role in the triggering of diabetic cardiomyopathy in DM2.

Cardiac O-GlcNAc signaling is increased in hypertrophy and heart failure

Reversible protein O-GlcNAc modification has emerged as an essential intracellular signaling system in several tissues, including cardiovascular pathophysiology related to diabetes and acute ischemic stress. We tested the hypothesis that cardiac O-GlcNAc signaling is altered in chronic cardiac hypertrophy and failure of different etiologies. Global protein O-GlcNAcylation and the main enzymes regulating O-GlcNAc, O-GlcNAc transferase (OGT), O-GlcNAcase (OGA), and glutamine-fructose-6-phosphate amidotransferase (GFAT) were measured by immunoblot and/or real-time RT-PCR analyses of left ventricular tissue from aortic stenosis (AS) patients and rat models of hypertension, myocardial infarction (MI), and aortic banding (AB), with and without failure. We show here that global O-GlcNAcylation was increased by 65% in AS patients, by 47% in hypertensive rats, by 81 and 58% post-AB, and 37 and 60% post-MI in hypertrophic and failing hearts, respectively (P < 0.05). Noticeably, protein O-GlcNAcylation patterns varied in hypertrophic vs. failing hearts, and the most extensive O-GlcNAcylation was observed on proteins of 20-100 kDa in size. OGT, OGA, and GFAT2 protein and/or mRNA levels were increased by pressure overload, while neither was regulated by myocardial infarction. Pharmacological inhibition of OGA decreased cardiac contractility in post-MI failing hearts, demonstrating a possible role of O-GlcNAcylation in development of chronic cardiac dysfunction. Our data support the novel concept that O-GlcNAc signaling is altered in various etiologies of cardiac hypertrophy and failure, including human aortic stenosis. This not only provides an exciting basis for discovery of new mechanisms underlying pathological cardiac remodeling but also implies protein O-GlcNAcylation as a possible new therapeutic target in heart failure.

The Hexosamine Biosyntheic Pathway and Platinum-Ethylenedicysteine-Glucosamine (Pt-ECG) for Targeted Therapy in Diffuse Large B-Cell Lymphoma

Abstract 587Aggressive non-Hodgkin lymphomas (NHL), such as diffuse large B cell lymphomas (DLBCL), are very common in the US with increasing incidences. Although these lymphomas are now potentially curable, almost half the treated patients still develop relapsed/refractory disease with poor survival outcomes, indicating an urgent need for better therapeutic approaches with improved efficacy.The hexosamine signaling pathway terminating in O-linked N-acetyl glucosamine (O-GlcNAc) cycling has been implicated in cellular signaling cascades and regulation of transcription factors involved in cancer biology. Biological functions of the hexosamine biosynthetic signaling pathways need elucidation, to determine whether altered O-GlcNAc metabolism plays a significant role in hematologic tumors such as DLBCL, and utilize this bifunctional pathway as a targeted therapeutic strategy in DLBCL. We have identified key enzymes of the hexosamine biosynthetic pathways to be highly-expressed in DLBCL cell lines and patient tumor cells. In contrast to normal circulating and tonsillar B cells, DLBCL cells expressed high levels of the rate limiting enzyme glutamine: fructose-6-phosphate amidotransferase (GFAT) as well as terminating enzyme O-GlcNAc transferase (OGT). We discovered that several key growth and survival transcription factors, such as NF-kB and NFAT, known to be highly-activated in DLBCL, are linked to the hexoasmine biosynthetic pathway. We demonstrated that both NF-kB (p65) and NFATc1 directly associated with OGT, and down-regulation of OGT by siRNA inhibits these transcription factors activation, suggesting that both NF-kB-p65 and NFATc1 require O-GlcNAc glycosylation by OGT for their activation. ChiP on Chip analysis on NFATc1 indicated that this transcription factor regulates a set of genes involved in glucose metabolism, including hexokinase and GFAT. These results suggest that the hexosamine pathway is highly active and utilized in DLBCL, and that exploiting this bi-functional pathway(s) as a therapeutic approach is feasible. We have previously developed an imaging agent, 99mTc-ethylenedicysteine-glucosamine (99mTc-EC-G) because EC-G mimics phosphorylated N-acetylglucosamine. ECG treatment in DLBCL cells enhances p65 and NFATc1 nuclear translocation. For therapeutic strategies, we developed metallic unlabeled Platinum (Pt) derivatives-EC-G as potential therapeutic agents. Pre-clinical in vitro studies have shown that our two lead compounds, Pt- and Pt-(DACH)-EC-G effectively inhibit lymphoma cell growth and induce apoptosis. These lead compounds can also induce DNA damage in DLBCL cells, through the up-regulation of phosphorylated histone 2AX (pH2AX), leading to the disruption of p65 and NFATc1 binding to DNA.This data importantly demonstrates that the hexosamine biosynthetic pathway is linked to key growth and survival pathways involved in the pathophysiology of DLBCL. Targeting these pathways with novel platinum EC-G compounds as a theranostic approach should lead to new, more effective treatments and diagnosis for DLBCL, particularly for relapsed/refractory DLBCL. Disclosures:Rollo:Cell Point: Employment.

Suppression of Glutamine:Fructose‐6‐phosphate amidotransferase‐1 inhibits adipogenesis in 3T3‐L1 adipocytes

O-linked N-acetylglucosamine (O-GlcNAc) protein modification has been implicated in the regulation of signaling pathways, cell function, and gene expression. Glutamine:fructose-6-phosphate amidotransferase-1 (GFAT-1) is the rate-limiting enzyme in the hexosamine biosynthetic pathway (HBP), which generates the sugar nucleotide UDP-GlcNAc, where this nucleotide acts as the donor for O-GlcNAc modification. In this study, we determined whether GFAT-1 regulates adipogenesis in adipocytes. 3T3-L1 preadipocytes were differentiated using medium containing high glucose, insulin, dexamethasone, and isobutylmethylxanthine. Cells were harvested 4, 8, and 12 h and 1, 2, 3, 4, 6, and 8 days after the initiation of differentiation. Global level of O-GlcNAc modification increased 4 h after induction and persisted for 8 days of observation. GFAT-1 mRNA and protein expression was also upregulated beginning 4 h after induction. Pharmacological inhibition of GFAT-1 or GFAT-1 siRNA treatment blocked the increase in O-GlcNAcylation and the formation of lipid droplets in adipocytes. GFAT-1 may regulate the expression of C/EBPβ, PPARγ, SREBP-1, fatty acid synthase, S3-12, perilipin, or adipophilin during adipogenesis. Our results suggest that GFAT-1 plays a critical role in modulating adipogenesis via the regulation of protein O-GlcNAcylation in adipocytes.

Discovery of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as glutamine fructose-6-phosphate amidotransferase (GFAT) inhibitors

Through high throughput screening and subsequent hit identification and optimization, we synthesized a series of 1-arylcarbonyl-6,7-dimethoxyisoquinoline derivatives as the first reported potent and reversible GFAT inhibitors. SAR studies of this class of compounds indicated significant impact on GFAT inhibition potency by substitutions on the A-ring and C-ring. The ketone group was found to be necessary for high potency. Compound 28 (RO0509347) demonstrated potent GFAT inhibition (IC(50)=1μM) with a desirable pharmacokinetic profile in rats, and showed significant efficacy in reducing the glucose excursion in an OGTT test in ob/ob mice.

Cellular Content of UDP-N-acetylhexosamines Controls Hyaluronan Synthase 2 Expression and Correlates with O-Linked N-Acetylglucosamine Modification of Transcription Factors YY1 and SP1

Hyaluronan, a high molecular mass polysaccharide on the vertebrate cell surface and extracellular matrix, is produced at the plasma membrane by hyaluronan synthases using UDP-GlcNAc and UDP-GlcUA as substrates. The availability of these UDP-sugar substrates can limit the synthesis rate of hyaluronan. In this study, we show that the cellular level of UDP-HexNAc also controls hyaluronan synthesis by modulating the expression of HAS2 (hyaluronan synthase 2). Increasing UDP-HexNAc in HaCaT keratinocytes by adding glucosamine down-regulated HAS2 gene expression, whereas a decrease in UDP-HexNAc, realized by mannose treatment or siRNA for GFAT1 (glutamine:fructose-6-phosphate amidotransferase 1), enhanced expression of the gene. Tracing the UDP-HexNAc-initiated signal to the HAS2 promoter revealed no change in the binding of STAT3, NF-κB, and cAMP response element-binding protein, shown previously to mediate growth factor and cytokine signals on HAS2 expression. Instead, altered binding of SP1 and YY1 to the promoter correlated with cellular UDP-HexNAc content and inhibition of HAS2 expression. siRNA silencing of YY1 and SP1 confirmed their inhibitory effects on HAS2 expression. Reduced and increased levels of O-GlcNAc-modified SP1 and YY1 proteins were associated with stimulation or inhibition of HAS2 expression, respectively. Our data are consistent with the hypothesis that, by regulating the level of protein O-GlcNAc modifications, cellular UDP-HexNAc content controls HAS2 transcription and decreases the effects on hyaluronan synthesis that would result from cellular fluctuations of this substrate.

Pleiotropic and Age-dependent Effects of Decreased Protein Modification by O-Linked N-Acetylglucosamine on Pancreatic β-Cell Function and Vascularization

The hexosamine biosynthesis pathway (HBP) regulates the post-translational modification of nuclear and cytoplasmic protein by O-linked N-acetylglucosamine (O-GlcNAc). Numerous studies have demonstrated increased flux through this pathway contributes to the development of β-cell dysfunction. The effect of decreased O-GlcNAc on the maintenance of normal β-cell function, however, is not well understood. We studied transgenic mice that over express β-N-acetylglucosaminidase (O-GlcNAcase), an enzyme that catalyzes the removal of O-GlcNAc from proteins, in the pancreatic β-cell under control of the rat insulin promoter. 3-4-Month-old O-GlcNAcase transgenic mice have higher glucose excursions with a concomitant decrease in circulating insulin levels, insulin mRNA levels, and total islet insulin content. In older (8-9-month-old) O-GlcNAcase transgenic mice glucose tolerance is no longer impaired. This is associated with increased serum insulin, islet insulin content, and insulin mRNA in the O-GlcNAcase transgenic mice. These improvements in β-cell function with aging are associated with increased angiogenesis and increased VEGF expression, with parallel increases in activation of Akt and expression of PGC1α. The biphasic effects as a function of age are consistent with published observations of mice with increased O-GlcNAc in islets and demonstrate that O-GlcNAc signaling exerts multiple effects on both insulin secretion and islet survival.

Evidence Coupling Increased Hexosamine Biosynthesis Pathway Activity to Membrane Cholesterol Toxicity and Cortical Filamentous Actin Derangement Contributing to Cellular Insulin Resistance†

Hyperinsulinemia is known to promote the progression/worsening of insulin resistance. Evidence reveals a hidden cost of hyperinsulinemia on plasma membrane (PM) phosphatidylinositol 4,5-bisphosphate (PIP(2))-regulated filamentous actin (F-actin) structure, components critical to the normal operation of the insulin-regulated glucose transport system. Here we delineated whether increased glucose flux through the hexosamine biosynthesis pathway (HBP) causes PIP(2)/F-actin dysregulation and subsequent insulin resistance. Increased glycosylation events were detected in 3T3-L1 adipocytes cultured under conditions closely resembling physiological hyperinsulinemia (5 nm insulin; 12 h) and in cells in which HBP activity was amplified by 2 mm glucosamine (GlcN). Both the physiological hyperinsulinemia and experimental GlcN challenge induced comparable losses of PIP(2) and F-actin. In addition to protecting against the insulin-induced membrane/cytoskeletal abnormality and insulin-resistant state, exogenous PIP(2) corrected the GlcN-induced insult on these parameters. Moreover, in accordance with HBP flux directly weakening PIP(2)/F-actin structure, pharmacological inhibition of the rate-limiting HBP enzyme [glutamine-fructose-6-phosphate amidotransferase (GFAT)] restored PIP(2)-regulated F-actin structure and insulin responsiveness. Conversely, overexpression of GFAT was associated with a loss of detectable PM PIP(2) and insulin sensitivity. Even less invasive challenges with glucose, in the absence of insulin, also led to PIP(2)/F-actin dysregulation. Mechanistically we found that increased HBP activity increased PM cholesterol, the removal of which normalized PIP(2)/F-actin levels. Accordingly, these data suggest that glucose transporter-4 functionality, dependent on PIP(2) and/or F-actin status, can be critically compromised by inappropriate HBP activity. Furthermore, these data are consistent with the PM cholesterol accrual/toxicity as a mechanistic basis of the HBP-induced defects in PIP(2)/F-actin structure and impaired glucose transporter-4 regulation.

Hexosamine Biosynthetic Pathway Mutations Cause Neuromuscular Transmission Defect

Neuromuscular junctions (NMJs) are synapses that transmit impulses from motor neurons to skeletal muscle fibers leading to muscle contraction. Study of hereditary disorders of neuromuscular transmission, termed congenital myasthenic syndromes (CMS), has helped elucidate fundamental processes influencing development and function of the nerve-muscle synapse. Using genetic linkage, we find 18 different biallelic mutations in the gene encoding glutamine-fructose-6-phosphate transaminase 1 (GFPT1) in 13 unrelated families with an autosomal recessive CMS. Consistent with these data, downregulation of the GFPT1 ortholog gfpt1 in zebrafish embryos altered muscle fiber morphology and impaired neuromuscular junction development. GFPT1 is the key enzyme of the hexosamine pathway yielding the amino sugar UDP-N-acetylglucosamine, an essential substrate for protein glycosylation. Our findings provide further impetus to study the glycobiology of NMJ and synapses in general.