Selective utilization of glucose metabolism guides mammalian gastrulation.

The prevailing dogma for morphological patterning in developing organisms argues that the combined inputs of transcription factor networks and signalling morphogens alone generate spatially and temporally distinct expression patterns. However, metabolism has also emerged as a critical developmental regulator1-10, independent of its functions in energy production and growth. The mechanistic role of nutrient utilization in instructing cellular programmes to shape the in vivo developing mammalian embryo remains unknown. Here we reveal two spatially resolved, cell-type- and stage-specific waves of glucose metabolism during mammalian gastrulation by using single-cell-resolution quantitative imaging of developing mouse embryos, stem cell models and embryo-derived tissue explants. We identify that the first spatiotemporal wave of glucose metabolism occurs through the hexosamine biosynthetic pathway to drive fate acquisition in the epiblast, and the second wave uses glycolysis to guide mesoderm migration and lateral expansion. Furthermore, we demonstrate that glucose exerts its influence on these developmental processes through cellular signalling pathways, with distinct mechanisms connecting glucose with the ERK activity in each wave. Our findings underscore that-in synergy with genetic mechanisms and morphogenic gradients-compartmentalized cellular metabolism is integral in guiding cell fate and specialized functions during development. This study challenges the view of the generic and housekeeping nature of cellular metabolism, offering valuable insights into its roles in various developmental contexts.

Phosphorylation of muramyl peptides by NAGK is required for NOD2 activation.

Bacterial cell wall components provide various unique molecular structures that are detected by pattern recognition receptors (PRRs) of the innate immune system as non-self. Most bacterial species form a cell wall that consists of peptidoglycan (PGN), a polymeric structure comprising alternating amino sugars that form strands cross-linked by short peptides. Muramyl dipeptide (MDP) has been well documented as a minimal immunogenic component of peptidoglycan1-3. MDP is sensed by the cytosolic nucleotide-binding oligomerization domain-containing protein 24 (NOD2). Upon engagement, it triggers pro-inflammatory gene expression, and this functionality is of critical importance in maintaining a healthy intestinal barrier function5. Here, using a forward genetic screen to identify factors required for MDP detection, we identified N-acetylglucosamine kinase (NAGK) as being essential for the immunostimulatory activity of MDP. NAGK is broadly expressed in immune cells and has previously been described to contribute to the hexosamine biosynthetic salvage pathway6. Mechanistically, NAGK functions upstream of NOD2 by directly phosphorylating the N-acetylmuramic acid moiety of MDP at the hydroxyl group of its C6 position, yielding 6-O-phospho-MDP. NAGK-phosphorylated MDP-but not unmodified MDP-constitutes an agonist for NOD2. Macrophages from mice deficient in NAGK are completely deficient in MDP sensing. These results reveal a link between amino sugar metabolism and innate immunity to bacterial cell walls.

Glucose influences endometrial receptivity to embryo implantation through O-GlcNAcylation-mediated regulation of the cytoskeleton.

Phenotypic changes to endometrial epithelial cells underpin receptivity to embryo implantation at the onset of pregnancy but the effect of hyperglycemia on these processes remains poorly understood. Here, we show that physiological levels of glucose (5 mM) abolished receptivity in the endometrial epithelial cell line, Ishikawa. However, embryo attachment was supported by 17 mM glucose as a result of glucose flux through the hexosamine biosynthetic pathway (HBP) and modulation of cell function via protein O-GlcNAcylation. Pharmacological inhibition of HBP or protein O-GlcNAcylation reduced embryo attachment in cocultures at 17 mM glucose. Mass spectrometry analysis of the O-GlcNAcylated proteome in Ishikawa cells revealed that myosin phosphatase target subunit 1 (MYPT1) is more highly O-GlcNAcylated in 17 mM glucose, correlating with loss of its target protein, phospho-myosin light chain 2, from apical cell junctions of polarized epithelium. Two-dimensional (2-D) and three-dimensional (3-D) morphologic analysis demonstrated that the higher glucose level attenuates epithelial polarity through O-GlcNAcylation. Inhibition of Rho (ras homologous)A-associated kinase (ROCK) or myosin II led to reduced polarity and enhanced receptivity in cells cultured in 5 mM glucose, consistent with data showing that MYPT1 acts downstream of ROCK signaling. These data implicate regulation of endometrial epithelial polarity through RhoA signaling upstream of actomyosin contractility in the acquisition of endometrial receptivity. Glucose levels impinge on this pathway through O-GlcNAcylation of MYPT1, which may impact endometrial receptivity to an implanting embryo in women with diabetes.NEW & NOTEWORTHY Understanding how glucose regulates endometrial function will support preconception guidance and/or the development of targeted interventions for individuals living with diabetes wishing to embark on pregnancy. We found that glucose can influence endometrial epithelial cell receptivity to embryo implantation by regulating posttranslational modification of proteins involved in the maintenance of cell polarity. Impaired or inappropriate endometrial receptivity could contribute to fertility and/or early pregnancy complications caused by poor glucose control.

TRIM14 suppressed the progression of NSCLC via hexosamine biosynthesis pathway.

Tripartite Motif 14 (TRIM14) is an oncoprotein that belongs to the E3 ligase TRIM family, which is involved in the progression of various tumors except for non-small cell lung carcinoma (NSCLC). However, little is currently known regarding the function and related mechanisms of TRIM14 in NSCLC. Here, we found that the TRIM14 protein was downregulated in lung adenocarcinoma tissues compared with the adjacent tissues, which can suppress tumor cell proliferation and migration both in vitro and in vivo. Moreover, TRIM14 can directly bind to glutamine fructose-6-phosphate amidotransferase 1 (GFAT1), which in turn results in the degradation of GFAT1 and reduced O-glycosylation levels. GFAT1 is a key enzyme in the rate-limiting step of the hexosamine biosynthetic pathway (HBP). Replenishment of N-acetyl-d-glucosamine can successfully reverse the inhibitory effect of TRIM14 on the NSCLC cell growth and migration as expected. Collectively, our data revealed that TRIM14 suppressed NSCLC cell proliferation and migration through ubiquitination and degradation of GFAT1, providing a new regulatory role for TRIM14 on HBP.

Infection-driven activation of transglutaminase 2 boosts glucose uptake and hexosamine biosynthesis in epithelial cells.

Transglutaminase 2 (TG2) is a ubiquitously expressed enzyme with transamidating activity. We report here that both expression and activity of TG2 are enhanced in mammalian epithelial cells infected with the obligate intracellular bacteria Chlamydia trachomatis. Genetic or pharmacological inhibition of TG2 impairs bacterial development. We show that TG2 increases glucose import by up-regulating the transcription of the glucose transporter genes GLUT-1 and GLUT-3. Furthermore, TG2 activation drives one specific glucose-dependent pathway in the host, i.e., hexosamine biosynthesis. Mechanistically, we identify the glucosamine:fructose-6-phosphate amidotransferase (GFPT) among the substrates of TG2. GFPT modification by TG2 increases its enzymatic activity, resulting in higher levels of UDP-N-acetylglucosamine biosynthesis and protein O-GlcNAcylation. The correlation between TG2 transamidating activity and O-GlcNAcylation is disrupted in infected cells because host hexosamine biosynthesis is being exploited by the bacteria, in particular to assist their division. In conclusion, our work establishes TG2 as a key player in controlling glucose-derived metabolic pathways in mammalian cells, themselves hijacked by C. trachomatis to sustain their own metabolic needs.

mTORC2 Responds to Glutamine Catabolite Levels to Modulate the Hexosamine Biosynthesis Enzyme GFAT1.

Highly proliferating cells are particularly dependent on glucose and glutamine for bioenergetics and macromolecule biosynthesis. The signals that respond to nutrient fluctuations to maintain metabolic homeostasis remain poorly understood. Here, we found that mTORC2 is activated by nutrient deprivation due to decreasing glutamine catabolites. We elucidate how mTORC2 modulates a glutamine-requiring biosynthetic pathway, the hexosamine biosynthesis pathway (HBP) via regulation of expression of glutamine:fructose-6-phosphate amidotransferase 1 (GFAT1), the rate-limiting enzyme of the HBP. GFAT1 expression is dependent on sufficient amounts of glutaminolysis catabolites particularly α-ketoglutarate, which are generated in an mTORC2-dependent manner. Additionally, mTORC2 is essential for proper expression and nuclear accumulation of the GFAT1 transcriptional regulator, Xbp1s. Thus, while mTORC1 senses amino acid abundance to promote anabolism, mTORC2 responds to declining glutamine catabolites in order to restore metabolic homeostasis. Our findings uncover the role of mTORC2 in metabolic reprogramming and have implications for understanding insulin resistance and tumorigenesis.

The kinase Isr1 negatively regulates hexosamine biosynthesis in S. cerevisiae.

The S. cerevisiae ISR1 gene encodes a putative kinase with no ascribed function. Here, we show that Isr1 acts as a negative regulator of the highly-conserved hexosamine biosynthesis pathway (HBP), which converts glucose into uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the carbohydrate precursor to protein glycosylation, GPI-anchor formation, and chitin biosynthesis. Overexpression of ISR1 is lethal and, at lower levels, causes sensitivity to tunicamycin and resistance to calcofluor white, implying impaired protein glycosylation and reduced chitin deposition. Gfa1 is the first enzyme in the HBP and is conserved from bacteria and yeast to humans. The lethality caused by ISR1 overexpression is rescued by co-overexpression of GFA1 or exogenous glucosamine, which bypasses GFA1's essential function. Gfa1 is phosphorylated in an Isr1-dependent fashion and mutation of Isr1-dependent sites ameliorates the lethality associated with ISR1 overexpression. Isr1 contains a phosphodegron that is phosphorylated by Pho85 and subsequently ubiquitinated by the SCF-Cdc4 complex, largely confining Isr1 protein levels to the time of bud emergence. Mutation of this phosphodegron stabilizes Isr1 and recapitulates the overexpression phenotypes. As Pho85 is a cell cycle and nutrient responsive kinase, this tight regulation of Isr1 may serve to dynamically regulate flux through the HBP and modulate how the cell's energy resources are converted into structural carbohydrates in response to changing cellular needs.

First characterization of glucose flux through the hexosamine biosynthesis pathway (HBP) in ex vivo mouse heart.

The hexosamine biosynthesis pathway (HBP) branches from glycolysis and forms UDP-GlcNAc, the moiety for O-linked ß-GlcNAc (O-GlcNAc) post-translational modifications. An inability to directly measure HBP flux has hindered our understanding of the factors regulating protein O-GlcNAcylation. Our goals in this study were to (i) validate a LC-MS method that assesses HBP flux as UDP-GlcNAc (13C)-molar percent enrichment (MPE) and concentration and (ii) determine whether glucose availability or workload regulate cardiac HBP flux. For (i), we perfused isolated murine working hearts with [U-13C6]glucosamine (1, 10, 50, or 100 µm), which bypasses the rate-limiting HBP enzyme. We observed a concentration-dependent increase in UDP-GlcNAc levels and MPE, with the latter reaching a plateau of 56.3 ± 2.9%. For (ii), we perfused isolated working hearts with [U-13C6]glucose (5.5 or 25 mm). Glycolytic efflux doubled with 25 mm [U-13C6]glucose; however, the calculated HBP flux was similar among the glucose concentrations at ∼2.5 nmol/g of heart protein/min, representing ∼0.003-0.006% of glycolysis. Reducing cardiac workload in beating and nonbeating Langendorff perfusions had no effect on the calculated HBP flux at ∼2.3 and 2.5 nmol/g of heart protein/min, respectively. To the best of our knowledge, this is the first direct measurement of glucose flux through the HBP in any organ. We anticipate that these methods will enable foundational analyses of the regulation of HBP flux and protein O-GlcNAcylation. Our results suggest that in the healthy ex vivo perfused heart, HBP flux does not respond to acute changes in glucose availability or cardiac workload.

Paramyxovirus replication induces the hexosamine biosynthetic pathway and mesenchymal transition via the IRE1α-XBP1s arm of the unfolded protein response.

The paramyxoviridae, respiratory syncytial virus (RSV), and murine respirovirus are enveloped, negative-sense RNA viruses that are the etiological agents of vertebrate lower respiratory tract infections (LRTIs). We observed that RSV infection in human small airway epithelial cells induced accumulation of glycosylated proteins within the endoplasmic reticulum (ER), increased glutamine-fructose-6-phosphate transaminases (GFPT1/2) and accumulation of uridine diphosphate (UDP)-N-acetylglucosamine, indicating activation of the hexosamine biosynthetic pathway (HBP). RSV infection induces rapid formation of spliced X-box binding protein 1 (XBP1s) and processing of activating transcription factor 6 (ATF6). Using pathway selective inhibitors and shRNA silencing, we find that the inositol-requiring enzyme (IRE1α)-XBP1 arm of the unfolded protein response (UPR) is required not only for activation of the HBP, but also for expression of mesenchymal transition (EMT) through the Snail family transcriptional repressor 1 (SNAI1), extracellular matrix (ECM)-remodeling proteins fibronectin (FN1), and matrix metalloproteinase 9 (MMP9). Probing RSV-induced open chromatin domains by ChIP, we find XBP1 binds and recruits RNA polymerase II to the IL6, SNAI1, and MMP9 promoters and the intragenic superenhancer of glutamine-fructose-6-phosphate transaminase 2 (GFPT2). The UPR is sustained through RSV by an autoregulatory loop where XBP1 enhances Pol II binding to its own promoter. Similarly, we investigated the effects of murine respirovirus infection on its natural host (mouse). Murine respirovirus induces mucosal growth factor response, EMT, and the indicators of ECM remodeling in an IRE1α-dependent manner, which persists after viral clearance. These data suggest that IRE1α-XBP1s arm of the UPR pathway is responsible for paramyxovirus-induced metabolic adaptation and mucosal remodeling via EMT and ECM secretion.

Cardiomyocyte protein O-GlcNAcylation is regulated by GFAT1 not GFAT2.

In response to cardiac injury, increased activity of the hexosamine biosynthesis pathway (HBP) is linked with cytoprotective as well as adverse effects depending on the type and duration of injury. Glutamine-fructose amidotransferase (GFAT; gene name gfpt) is the rate-limiting enzyme that controls flux through HBP. Two protein isoforms exist in the heart called GFAT1 and GFAT2. There are conflicting data on the relative importance of GFAT1 and GFAT2 during stress-induced HBP responses in the heart. Using neonatal rat cardiac cell preparations, targeted knockdown of GFPT1 and GFPT2 were performed and HBP activity measured. Immunostaining with specific GFAT1 and GFAT2 antibodies was undertaken in neonatal rat cardiac preparations and murine cardiac tissues to characterise cell-specific expression. Publicly available human heart single cell sequencing data was interrogated to determine cell-type expression. Western blots for GFAT isoform protein expression were performed in human cardiomyocytes derived from induced pluripotent stem cells (iPSCs). GFPT1 but not GFPT2 knockdown resulted in a loss of stress-induced protein O-GlcNAcylation in neonatal cardiac cell preparations indicating reduced HBP activity. In rodent cells and tissue, immunostaining for GFAT1 identified expression in both cardiac myocytes and fibroblasts whereas immunostaining for GFAT2 was only identified in fibroblasts. Further corroboration of findings in human heart cells identified an enrichment of GFPT2 gene expression in cardiac fibroblasts but not ventricular myocytes whereas GFPT1 was expressed in both myocytes and fibroblasts. In human iPSC-derived cardiomyocytes, only GFAT1 protein was expressed with an absence of GFAT2. In conclusion, these results indicate that GFAT1 is the primary cardiomyocyte isoform and GFAT2 is only present in cardiac fibroblasts. Cell-specific isoform expression may have differing effects on cell function and should be considered when studying HBP and GFAT functions in the heart.

Insulin resistance in glomerular podocytes: Potential mechanisms of induction.

Glomerular podocytes are a target for the actions of insulin. Accumulating evidence indicates that exposure to nutrient overload induces insulin resistance in these cells, manifested by abolition of the stimulatory effect of insulin on glucose uptake. Numerous recent studies have investigated potential mechanisms of the induction of insulin resistance in podocytes. High glucose concentrations stimulated reactive oxygen species production through NADPH oxidase activation, decreased adenosine monophosphate-activated protein kinase (AMPK) phosphorylation, and reduced deacetylase sirtuin 1 (SIRT1) protein levels and activity. Calcium signaling involving transient receptor potential cation channel C, member 6 (TRPC6) also was demonstrated to play an essential role in the regulation of insulin-dependent signaling and glucose uptake in podocytes. Furthermore, podocytes exposed to diabetic environment, with elevated insulin levels become insulin resistant as a result of degradation of insulin receptor (IR), resulting in attenuation of insulin signaling responsiveness. Also elevated levels of palmitic acid appear to be an important factor and contributor to podocytes insulin resistance. This review summarizes cellular and molecular alterations that contribute to the development of insulin resistance in glomerular podocytes.

Inhibiting the Hexosamine Biosynthetic Pathway Lowers O-GlcNAcylation Levels and Sensitizes Cancer to Environmental Stress.

The amounts of the intracellular glycosylation, O-GlcNAc modification, are increased in essentially all tumors when compared to healthy tissue, and lowering O-GlcNAcylation levels results in reduced tumorigenesis and increased cancer cell death. Therefore, the pharmacological reduction of O-GlcNAc may represent a therapeutic vulnerability. The most direct approach to this goal is the inhibition of O-GlcNAc transferase (OGT), the enzyme that directly adds the modification to proteins. However, despite some recent success, this enzyme has proven difficult to inhibit. An alternative strategy involves starving OGT of its sugar substrate UDP-GlcNAc by targeting enzymes of the hexosamine biosynthetic pathway (HBP). Here, we explore the potential of the rate-determining enzyme of this pathway, glutamine fructose-6-phosphate amidotransferase (GFAT). We first show that CRISPR-mediated knockout of GFAT results in inhibition of cancer cell growth in vitro and a xenograft model that correlates with O-GlcNAcylation levels. We then demonstrate that pharmacological inhibition of GFAT sensitizes a small panel of cancer cells to undergo apoptosis in response to diamide-induced oxidative stress. Finally, we find that GFAT expression and O-GlcNAc levels are increased in a spontaneous mouse model of liver cancer. Together these experiments support the further development of inhibitors of the HBP as an indirect approach to lowering O-GlcNAcylation levels in cancer.

The role of O-GlcNAcylation in immunity against infections.

Mounting an effective immune response is crucial for the host to protect itself against invading pathogens. It is now well appreciated that reprogramming of core metabolic pathways in immune cells is a key requirement for their activation and function during infections. The role of several ancillary metabolic pathways in shaping immune cell function is less well understood. One such pathway, for which interest has recently been growing, is the hexosamine biosynthesis pathway (HBP) that generates uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the donor substrate for a specific form of glycosylation termed O-GlcNAcylation. O-GlcNAc is an intracellular post-translational modification that alters the functional properties of the modified proteins, in particular transcription factors and epigenetic regulators. An increasing number of studies suggest a central role for the HBP and O-GlcNAcylation in dictating immune cell function, including the response to different pathogens. We here discuss the most recent insights regarding O-GlcNAcylation and immunity, and explore whether targeting of O-GlcNAcylation could hold promise as a therapeutic approach to modulate immune responses to infections.

Probiotic, Bacillus subtilis E20 alters the immunity of white shrimp, Litopenaeus vannamei via glutamine metabolism and hexosamine biosynthetic pathway.

The purpose of this study was to profile the mechanisms of action of probiotic, Bacillus subtilis E20 in activating the immunity of white shrimp, Litopenaeus vannamei. Two groups of shrimp were studied. One group was fed a control diet without probiotic supplementation and the other was fed a probiotic-containing diet at a level of 109 cfu kg diet-1. After the 8-week feeding regimen, the metabolite composition in the hepatopancreas of shrimp were investigated using 1H nuclear magnetic resonance (1H NMR) based metabolomic analysis. Results from the 1H NMR analysis revealed that 16 hepatopancreatic metabolites were matched and identified among groups, of which 2 metabolites, creatinine and glutamine were significantly higher in probiotic group than in the control group. This result was confirmed by the reverse-phase high-performance liquid chromatography (RP-HPLC) and spectrophotometric analysis. Transcriptome analysis indicated the expressions of 10 genes associated with antioxidant enzymes, pattern recognition proteins and antimicrobial molecules, more active expression in the shrimp fed a diet supplemented with probiotic as compared to that of shrimp in control. In addition, the expressions of 4 genes involved with hexosamine biosynthesis pathway (HBP) and UDP-N-acetylglucosamine-peptide N-acetylglucosaminyltransferase for protein O-glycosylation were also higher in hepatopancreas of probiotic-treated shrimp than in shrimp fed a control diet. Western blot and enzyme-linked immunosorbent assay showed that heat shock factor 1, heat shock protein 70, and protein O-glycosylation in hepatopancreas were higher in probiotic group than the control group. These findings suggest that probiotic, B. subtilis E20 promotes the digestibility of glutamine in the diet, and that the increased glutamine in shrimp can be used as fuel for immune cells or may be used to regulate immune molecule expressions and protein O-glycosylation via the HBP to increase protein O-glycosylation, thereby improving the health of shrimp.

Role of the O-GlcNAc modification on insulin resistance and endoplasmic reticulum stress in 3T3-L1 cells.

O-linked N-acetyl-glucosamine (O-GlcNAc) is a post-translational protein modification that regulates cell signaling and involves in several pathological conditions. O-GlcNAc transferase (OGT) catalyzes the attachment, while O-GlcNAcase (OGA) splits the GlcNAc molecules from the serine or threonine residues of the nuclear and cellular proteins. The hexosamine biosynthesis pathway (HBP) is a small branch of glycolysis that provides a substrate for the OGT and serves as a nutrient sensor. In this study, we investigated the impact of external O-GlcNAc modification stimulus on the insulin signal transduction, unfolded protein response, and HBP in 3T3-L1 cells. First, we treated cells with glucosamine and PUGNAc to stimulate the O-GlcNAcylation of total proteins. Also, we treated cells with tunicamycin as a positive internal control, which is a widely-used endoplasmic reticulum stressor. We used two in vitro models to understand the impact of the cellular state of insulin sensibility on this hypothesis. So, we employed insulin-sensitive preadipocytes and insulin-resistant adipocytes to answer these questions. Secondly, the OGT-silencing achieved in the insulin-resistant preadipocyte model by using the short-hairpin RNA (shRNA) interference method. Thereafter, the cells treated with the above-mentioned compounds to understand the role of the diminished O-GlcNAc protein modification on the insulin signal transduction, unfolded protein response, and HBP. We found that elevated O-GlcNAcylation of the total proteins displayed a definite correlation in insulin resistance and endoplasmic reticulum stress. Furthermore, we identified that the degree of this correlation depends on the cellular state of insulin sensitivity. Moreover, reduced O-GlcNAcylation of the total proteins by the shRNA-mediated silencing of the OGT gene, which is the only gene to modify proteins with the O-GlcNAc molecule, reversed the insulin resistance and endoplasmic reticulum stress phenotype, even with the externally stimulated O-GlcNAc modification conditions. In conclusion, our results suggest that OGT regulates insulin receptor signaling and unfolded protein response by modulating O-GlcNAc levels of total proteins, in response to insulin resistance. Therefore, it can be a potential therapeutic target to prevent insulin resistance and endoplasmic reticulum stress.

mTORC2 modulates the amplitude and duration of GFAT1 Ser-243 phosphorylation to maintain flux through the hexosamine pathway during starvation.

The mechanistic target of rapamycin (mTOR) controls metabolic pathways in response to nutrients. Recently, we have shown that mTOR complex 2 (mTORC2) modulates the hexosamine biosynthetic pathway (HBP) by promoting the expression of the key enzyme of the HBP, glutamine:fructose-6-phosphate aminotransferase 1 (GFAT1). Here, we found that GFAT1 Ser-243 phosphorylation is also modulated in an mTORC2-dependent manner. In response to glutamine limitation, active mTORC2 prolongs the duration of Ser-243 phosphorylation, albeit at lower amplitude. Blocking glycolysis using 2-deoxyglucose robustly enhances Ser-243 phosphorylation, correlating with heightened mTORC2 activation, increased AMPK activity, and O-GlcNAcylation. However, when 2-deoxyglucose is combined with glutamine deprivation, GFAT1 Ser-243 phosphorylation and mTORC2 activation remain elevated, whereas AMPK activation and O-GlcNAcylation diminish. Phosphorylation at Ser-243 promotes GFAT1 expression and production of GFAT1-generated metabolites including ample production of the HBP end-product, UDP-GlcNAc, despite nutrient starvation. Hence, we propose that the mTORC2-mediated increase in GFAT1 Ser-243 phosphorylation promotes flux through the HBP to maintain production of UDP-GlcNAc when nutrients are limiting. Our findings provide insights on how the HBP is reprogrammed via mTORC2 in nutrient-addicted cancer cells.

Inhibition of the hexosamine biosynthesis pathway potentiates cisplatin cytotoxicity by decreasing BiP expression in non-small-cell lung cancer cells.

Platinum anticancer agents are essential components in chemotherapeutic regimens for non-small-cell lung cancer (NSCLC) patients ineligible for targeted therapy. However, platinum-based regimens have reached a plateau of therapeutic efficacy; therefore, it is critical to implement novel approaches for improvement. The hexosamine biosynthesis pathway (HBP), which produces amino-sugar N-acetyl-glucosamine for protein glycosylation, is important for protein function and cell survival. Here we show a beneficial effect by the combination of cisplatin with HBP inhibition. Expression of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme of HBP, was increased in NSCLC cell lines and tissues. Pharmacological inhibition of GFAT activity or knockdown of GFATimpaired cell proliferation and exerted synergistic or additive cytotoxicity to the cells treated with cisplatin. Mechanistically, GFAT positively regulated the expression of binding immunoglobulin protein (BiP; also known as glucose-regulated protein 78, GRP78), an endoplasmic reticulum chaperone involved in unfolded protein response (UPR). Suppressing GFAT activity resulted in downregulation of BiP that activated inositol-requiring enzyme 1α, a sensor protein of UPR, and exacerbated cisplatin-induced cell apoptosis. These data identify GFAT-mediated HBP as a target for improving platinum-based chemotherapy for NSCLC.

Cellular glycosylation senses metabolic changes and modulates cell plasticity during epithelial to mesenchymal transition.

Epithelial to mesenchymal transition (EMT) is a developmental program reactivated by tumor cells that leads to the switch from epithelial to mesenchymal phenotype. During EMT, cells are transcriptionally regulated to decrease E-cadherin expression while expressing mesenchymal markers such as vimentin, fibronectin, and N-cadherin. Growing body of evidences suggest that cells engaged in EMT undergo a metabolic reprograming process, redirecting glucose flux toward hexosamine biosynthesis pathway (HBP), which fuels aberrant glycosylation patterns that are extensively observed in cancer cells. HBP depends on nutrient availability to produce its end product UDP-GlcNAc, and for this reason is considered a metabolic sensor pathway. UDP-GlcNAc is the substrate used for the synthesis of major types of glycosylation, including O-GlcNAc and cell surface glycans. In general, the rate limiting enzyme of HBP, GFAT, is overexpressed in many cancer types that present EMT features as well as aberrant glycosylation. Moreover, altered levels of O-GlcNAcylation can modulate cell morphology and favor EMT. In this review, we summarize some of the current knowledge that correlates glucose metabolism, aberrant glycosylation and hyper O-GlcNAcylation supported by HBP that leads to EMT activation. Developmental Dynamics 247:481-491, 2018. © 2017 Wiley Periodicals, Inc.

Central Role of Metabolism in Endothelial Cell Function and Vascular Disease.

The importance of endothelial cell (EC) metabolism and its regulatory role in the angiogenic behavior of ECs during vessel formation and in the function of different EC subtypes determined by different vascular beds has been recognized only in the last few years. Even more importantly, apart from a role of nitric oxide and reactive oxygen species in EC dysfunction, deregulations of EC metabolism in disease only recently received increasing attention. Although comprehensive metabolic characterization of ECs still needs further investigation, the concept of targeting EC metabolism to treat vascular disease is emerging. In this overview, we summarize EC-specific metabolic pathways, describe the current knowledge on their deregulation in vascular diseases, and give an outlook on how vascular endothelial metabolism can serve as a target to normalize deregulated endothelium.

"Nutrient-sensing" and self-renewal: O-GlcNAc in a new role.

Whether embryonic, hematopoietic or cancer stem cells, this metabolic reprogramming is dependent on the nutrient-status and bioenergetic pathways that is influenced by the micro-environmental niches like hypoxia. Thus, the microenvironment plays a vital role in determining the stem cell fate by inducing metabolic reprogramming. Under the influence of the microenvironment, like hypoxia, the stem cells have increased glucose and glutamine uptake which result in activation of hexosamine biosynthesis pathway (HBP) and increased O-GlcNAc Transferase (OGT). The current review is focused on understanding how HBP, a nutrient-sensing pathway (that leads to increased OGT activity) is instrumental in regulating self-renewal not only in embryonic and hematopoietic stem cells (ESC/HSC) but also in cancer stem cells.