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The potential use of pro-senescence therapies, known as TIS (Therapy-Induced Senescence), for the treatment of colorectal cancer (CRC) generated significant interest since they require lower doses compared to those required for inducing apoptosis. However, the senescent cell cycle-arrested cancer cells are long-lived, and studies have revealed escape mechanisms contributing to tumor recurrence. To deepen our understanding of the survival pathways used by senescent cancer cells, we delved into the potential involvement of the hexosamine biosynthetic pathway (HBP). HBP provides UDP-GlcNAc, the substrate for O-GlcNAc transferase (OGT), which catalyzes O-GlcNAcylation, a post-translational modification implicated in regulating numerous cellular functions and aberrantly elevated in CRC. In this study, we demonstrated, in the p53-proficient colon cancer cell lines HCT116 and LS174T, that TIS induced by low-dose SN38 or etoposide treatment was accompanied with a decrease of GFAT (the rate limiting enzyme of the HBP), OGT and O-GlcNAcase (OGA) expression correlated with a slight reduction in O-GlcNAcylation levels. Further decreasing this level of O-GlcNAcylation by knocking-down GFAT or OGT redirected the cellular response to subtoxic chemotherapy doses from senescence to apoptosis, in correlation with an enhancement of DNA damages. Pharmacological inhibition of OGT with OSMI-4 in HCT116 and LS174T cells and in a patient-derived colon tumoroid model supported these findings. Taken together, these results suggest that combing O-GlcNAcylation inhibitors to low doses of conventional chemotherapeutic drugs could potentially reduce treatment side effects while preserving efficacy. Furthermore, this approach may increase treatment specificity, as CRC cells exhibit higher O-GlcNAcylation levels compared to normal tissues., (© 2024. The Author(s).)

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 regulator 1-10 , 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., (© 2024. The Author(s).)

Age is a prominent risk factor for cardiometabolic disease, often leading to heart structural and functional changes. However, precise molecular mechanisms underlying cardiac remodeling and dysfunction exclusively resulting from physiological aging remain elusive. Previous research demonstrated age-related functional alterations in baboons, analogous to humans. The goal of this study is to identify early cardiac molecular alterations preceding functional adaptations, shedding light on the regulation of age-associated changes. Unbiased transcriptomics of left ventricle samples are performed from female baboons aged 7.5-22.1 years (human equivalent ≈30-88 years). Weighted-gene correlation network and pathway enrichment analyses are performed, with histological validation. Modules of transcripts negatively correlated with age implicated declined metabolism-oxidative phosphorylation, tricarboxylic acid cycle, glycolysis, and fatty-acid β-oxidation. Transcripts positively correlated with age suggested a metabolic shift toward glucose-dependent anabolic pathways, including hexosamine biosynthetic pathway (HBP). This shift is associated with increased glycosaminoglycan synthesis, modification, precursor synthesis via HBP, and extracellular matrix accumulation, verified histologically. Upregulated extracellular matrix-induced signaling coincided with glycosaminoglycan accumulation, followed by cardiac hypertrophy-related pathways. Overall, these findings revealed a transcriptional shift in metabolism favoring glycosaminoglycan accumulation through HBP before cardiac hypertrophy. Unveiling this metabolic shift provides potential targets for age-related cardiac diseases, offering novel insights into early age-related mechanisms., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Our previous studies revealed a novel link between gemcitabine (GEM) chemotherapy and elevated glutamine-fructose-6-phosphate transaminase 2 (GFPT2) expression in pancreatic cancer (PaCa) cells. GFPT2 is a rate-limiting enzyme in the hexosamine biosynthesis pathway (HBP). HBP can enhance metastatic potential by regulating epithelial-mesenchymal transition (EMT). The aim of this study was to further evaluate the effect of chemotherapy-induced GFPT2 expression on metastatic potential. GFPT2 expression was evaluated in a mouse xenograft model following GEM exposure and in clinical specimens of patients after chemotherapy using immunohistochemical analysis. The roles of GFPT2 in HBP activation, downstream pathways, and cellular functions in PaCa cells with regulated GFPT2 expression were investigated. GEM exposure increased GFPT2 expression in tumors resected from a mouse xenograft model and in patients treated with neoadjuvant chemotherapy (NAC). GFPT2 expression was correlated with post-operative liver metastasis after NAC. Its expression activated the HBP, promoting migration and invasion. Treatment with HBP inhibitors reversed these effects. Additionally, GFPT2 upregulated ZEB1 and vimentin expression and downregulated E-cadherin expression. GEM induction upregulated GFPT2 expression. Elevated GFPT2 levels promoted invasion by activating the HBP, suggesting the potential role of this mechanism in promoting chemotherapy-induced metastasis., (© 2024. The Author(s).)

There is a growing interest in the creation of engineered condensates formed via liquid-liquid phase separation (LLPS) to exert precise cellular control in prokaryotes. However, de novo design of cellular condensates to control metabolic flux or protein translation remains a challenge. Here, we present a synthetic condensate platform, generated through the incorporation of artificial, disordered proteins to realize specific functions in Bacillus subtilis. To achieve this, the "stacking blocks" strategy is developed to rationally design a series of LLPS-promoting proteins for programming condensates. Through the targeted recruitment of biomolecules, our investigation demonstrates that cellular condensates effectively sequester biosynthetic pathways. We successfully harness this capability to enhance the biosynthesis of 2'-fucosyllactose by 123.3%. Furthermore, we find that condensates can enhance the translation specificity of tailored enzyme fourfold, and can increase N-acetylmannosamine titer by 75.0%. Collectively, these results lay the foundation for the design of engineered condensates endowed with multifunctional capacities., (© 2024. The Author(s).)

UDP- N -acetylglucosamine pyrophosphorylase (UAP) catalyzes the last step in the hexosamine biosynthesis pathway to directly produce UDP- N -acetylglucosamine (UDP-GlcNAc). Because UAPs play important physiological and pathological roles in organisms, they are considered potential targets for drug and pesticide development. However, the lack of efficient and selective inhibitors is a bottleneck that must be overcome. This study reports the first crystal structure of the insect UAP from Spodoptera frugiperda ( Sf UAP) in complex with UDP-GlcNAc. Sf UAP has two insect-specific structural characteristics in the active pocket, namely, a free Cys (Cys 334 ) and a Mg 2+ binding site, which differentiate it from human UAP ( Hs AGX1) and fungal UAP ( Af UAP) in terms of substrate and inhibitor binding. N -(4-Nitrophenyl)maleimide ( p NPMI) and myricetin are discovered as potent covalent and noncovalent inhibitors of Sf UAP, respectively. Moreover, myricetin can significantly reduce the level of cellular O -GlcNAcylation by inhibiting both UAP and O -GlcNAc transferase. These findings provide novel insights into the development of UAP-based drugs and pesticides.

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.

Hepatocellular carcinoma (HCC) presents significant challenges in treatment and prognosis because of its aggressive nature and high metastatic potential. This study aims to investigate the role of the hexosamine biosynthesis pathway (HBP) and its association with HCC progression and prognosis. We identified SPP1 and FOXM1 as hub genes within the HBP pathway, showing their correlation with poor prognosis and late-stage progression. In addition, the analysis uncovered the complex participation of the HBP pathway in nutrients and oxygen reactions, PI3K-AKT signaling, AMPK activation, and angiogenesis regulation. The disruption of these pathways is pivotal in influencing the growth and progression of HCC. Targeting the HBP presents a promising therapeutic approach to modulate the tumor microenvironment, thereby enhancing the efficacy of immunotherapy. In addition, FOXM1 was identified as the HBP pathway regulator, influencing cellular O-GlcNAcylation level and VEGF secretion, thereby promoting angiogenesis in HCC. Inhibition of O-GlcNAcylation significantly hindered angiogenesis, which is suggested as a potential avenue for therapeutic intervention. Our research demonstrates the practicality of using the HBP-related gene as a prognostic marker in liver cancer patients and suggests targeting FOXM1 as a novel avenue for personalized therapy., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

CD36 is a type 2 cell surface scavenger receptor expressed in various tissues. In macrophages, CD36 recognizes oxidized low-density lipoprotein (ox-LDL), which promotes the formation of foam cells, the first step toward an atherosclerotic arterial lesion. CD36 possesses a variety of posttranslational modifications, among them N-glycosylation and O-GlcNAc modification. Some of the roles of these modifications on CD36 are known, such as N-linked glycosylation, which provides proper folding and trafficking to the plasma membrane in the human embryonic kidney. This study aimed to determine whether variations in the availability of UDP-GlcNAc could impact Rab-5-mediated endocytic trafficking and, therefore, the cellular localization of CD36. These preliminary results suggest that the availability of the substrate UDP-GlcNAc, modulated in response to treatment with Thiamet G (TMG), OSMI-1 (O-GlcNAcylation enzymes modulators) or Azaserine (HBP modulator), influences the localization of CD36 in J774 macrophages, and the endocytic trafficking as evidenced by the regulatory protein Rab-5, between the plasma membrane and the cytoplasm., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2024 Loaeza-Reyes, Zenteno, Ramírez-Hernández, Salinas-Marin, Moreno-Rodríguez, Torres-Rosas, Argueta-Figueroa, Fernández-Rojas, Pina-Canseco, Acevedo-Mascarúa, Hernández-Antonio and Pérez-Cervera.)

Disrupted glucose metabolism and protein misfolding are key characteristics of age-related neurodegenerative disorders including Parkinson's disease, however their mechanistic linkage is largely unexplored. The hexosamine biosynthetic pathway utilizes glucose and uridine-5'-triphosphate to generate N-linked glycans required for protein folding in the endoplasmic reticulum. Here we find that Parkinson's patient midbrain cultures accumulate glucose and uridine-5'-triphosphate, while N-glycan synthesis rates are reduced. Impaired glucose flux occurred by selective reduction of the rate-limiting enzyme, GFPT2, through disrupted signaling between the unfolded protein response and the hexosamine pathway. Failure of the unfolded protein response and reduced N-glycosylation caused immature lysosomal hydrolases to misfold and accumulate, while accelerating glucose flux through the hexosamine pathway rescued hydrolase function and reduced pathological α-synuclein. Our data indicate that the hexosamine pathway integrates glucose metabolism with lysosomal activity, and its failure in Parkinson's disease occurs by uncoupling of the unfolded protein response-hexosamine pathway axis. These findings offer new methods to restore proteostasis by hexosamine pathway enhancement., (© 2024. The Author(s).)

Cyanovirin-N (CV-N) binds high-mannose oligosaccharides on enveloped viruses with two carbohydrate-binding sites, one bearing high affinity and one low affinity to Manα(1-2)Man moieties. A tandem repeat of two CV-N molecules (CVN2) was tested for antiviral activity against human immunodeficiency virus type I (HIV-1) by using a domain-swapped dimer. CV-N was shown to bind N -acetylmannosamine (ManNAc) and N -acetyl-d-glucosamine (GlcNAc) when the carbohydrate-binding sites in CV-N were free to interact with these monosaccharides independently. CVN2 recognized ManNAc at a K d of 1.4 μM and bound this sugar in solution, regardless of the lectin making amino acid side chain contacts on the targeted viral glycoproteins. An interdomain cross-contacting residue Glu41, which has been shown to be hydrogen bonding with dimannose, was substituted in the monomeric CV-N. The amide derivative of glucose, GlcNAc, achieved similar high affinity to the new variant CVN-E41T as high-mannose N -glycans, but binding to CVN2 in the nanomolar range with four binding sites involved or binding to the monomeric CVN-E41A. A stable dimer was engineered and expressed from the alanine-to-threonine-substituted monomer to confirm binding to GlcNAc. In summary, low-affinity binding was achieved by CVN2 to dimannosylated peptide or GlcNAc with two carbohydrate-binding sites of differing affinities, mimicking biological interactions with the respective N -linked glycans of interest and cross-linking of carbohydrates on human T cells for lymphocyte activation.

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., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

Lung cancer is a highly prevalent malignancy characterized by significant metabolic alterations. Understanding the metabolic rewiring in lung cancer is crucial for the development of effective therapeutic strategies. The hexosamine biosynthesis pathway (HBP) is a metabolic pathway that plays a vital role in cellular metabolism and has been implicated in various cancers, including lung cancer. Abnormal activation of HBP is involved in the proliferation, progression, metastasis, and drug resistance of tumor cells. In this review, we will discuss the function and regulation of metabolic enzymes related to HBP in lung cancer. Furthermore, the implications of targeting the HBP for lung cancer treatment are also discussed, along with the challenges and future directions in this field. This review provides a comprehensive understanding of the role and intervention of HBP in lung cancer. Future research focusing on the HBP in lung cancer is essential to uncover novel treatment strategies and improve patient outcomes., (© 2023. BioMed Central Ltd., part of Springer Nature.)

Impaired brain glucose metabolism is considered a hallmark of brain dysfunction and neurodegeneration. Disruption of the hexosamine biosynthetic pathway (HBP) and subsequent O -linked N -acetylglucosamine ( O -GlcNAc) cycling has been identified as an emerging link between altered glucose metabolism and defects in the brain. Myriads of cytosolic and nuclear proteins in the nervous system are modified at serine or threonine residues with a single N -acetylglucosamine ( O -GlcNAc) molecule by O -GlcNAc transferase (OGT), which can be removed by β- N -acetylglucosaminidase ( O -GlcNAcase, OGA). Homeostatic regulation of O -GlcNAc cycling is important for the maintenance of normal brain activity. Although significant evidence linking dysregulated HBP metabolism and aberrant O -GlcNAc cycling to induction or progression of neuronal diseases has been obtained, the issue of whether altered O -GlcNAcylation is causal in brain pathogenesis remains uncertain. Elucidation of the specific functions and regulatory mechanisms of individual O -GlcNAcylated neuronal proteins in both normal and diseased states may facilitate the identification of novel therapeutic targets for various neuronal disorders. The information presented in this review highlights the importance of HBP/ O -GlcNAcylation in the neuronal system and summarizes the roles and potential mechanisms of O -GlcNAcylated neuronal proteins in maintaining normal brain function and initiation and progression of neurological diseases.

Purpose: In type 2 Diabetes, β-cell failure is caused by loss of cell mass, mostly by apoptosis, but also by simple dysfunction (dedifferentiation, decline of glucose-stimulated insulin secretion). Apoptosis and dysfunction are caused, at least in part, by glucotoxicity, in which increased flux of glucose in the hexosamine biosynthetic pathway plays a role. In this study, we sought to clarify whether increased hexosamine biosynthetic pathway flux affects another important aspect of β-cell physiology, that is β-cell-β-cell homotypic interactions., Methods: We used INS-1E cells and murine islets. The expression and cellular distribution of E-cadherin and β-catenin was evaluated by immunofluorescence, immunohistochemistry and western blot. Cell-cell adhesion was examined by the hanging-drop aggregation assay, islet architecture by isolation and microscopic observation., Results: E-cadherin expression was not changed by increased hexosamine biosynthetic pathway flux, however, there was a decrease of cell surface, and an increase in intracellular E-cadherin. Moreover, intracellular E-cadherin delocalized, at least in part, from the Golgi complex to the endoplasmic reticulum. Beta-catenin was found to parallel the E-cadherin redistribution, showing a dislocation from the plasmamembrane to the cytosol. These changes had as a phenotypic consequence a decreased ability of INS-1E to aggregate. Finally, in ex vivo experiments, glucosamine was able to alter islet structure and to decrease surface abundandance of E-cadherin and β-catenin., Conclusion: Increased hexosamine biosynthetic pathway flux alters E-cadherin cellular localization both in INS-1E cells and murine islets and affects cell-cell adhesion and islet morphology. These changes are likely caused by alterations of E-cadherin function, highlighting a new potential target to counteract the consequences of glucotoxicity on β-cells., (© 2023. The Author(s).)

The hexosamine biosynthesis pathway (HBP) is a glucose metabolism pathway that produces uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). Substantial changes in HBP, including elevated HBP flux and UDP-GlcNAc levels, are associated with cancer pathogenesis. Particularly, cancer cells expressing oncogenic Kirsten rat sarcoma virus (KRAS) are highly dependent on HBP for growth and survival. To differentiate between HBP metabolites in KRAS wild-type (WT) and mutant (MT) lung cancer cells, a simultaneous quantitative method for analyzing seven HPB metabolites was developed using ultra-high-performance liquid chromatography-tandem mass spectrometry. A simple method without complicated preparation steps, such as derivatization or isotope labeling, was optimized for the simultaneous analysis of highly hydrophilic HBP metabolites, and the developed method was successfully verified. The intra- and inter-day coefficients of variation were less than 15% for all HBP metabolites, and the recovery was 89.67-114.5%. All results of the validation list were in accordance with ICM M10 guidelines. Through this method, HBP metabolites in lung cancer cells were accurately quantified, and it was confirmed that all HBP metabolites were upregulated in KRAS MT cells compared with KRAS WT lung cancer cells. We expect that this will be a useful tool for metabolic research on cancer and for the development of new drugs for cancer treatment., (© 2023 John Wiley & Sons Ltd.)

Background: Diabetic retinopathy (DR) remains one of the most common complications of diabetes despite great efforts to uncover its underlying mechanisms. The pathogenesis of DR is characterized by the deterioration of the neurovascular unit (NVU), showing damage of vascular cells, activation of glial cells and dysfunction of neurons. Activation of the hexosamine biosynthesis pathway (HBP) and increased protein O-GlcNAcylation have been evident in the initiation of DR in patients and animal models., Scope of Review: The impairment of the NVU, in particular, damage of vascular pericytes and endothelial cells arises in hyperglycemia-independent conditions as well. Surprisingly, despite the lack of hyperglycemia, the breakdown of the NVU is similar to the pathology in DR, showing activated HBP, altered O-GlcNAc and subsequent cellular and molecular dysregulation., Major Conclusions: This review summarizes recent research evidence highlighting the significance of the HBP in the breakdown of the NVU in hyperglycemia-dependent and -independent manners, and thus identifies joint avenues leading to vascular damage as seen in DR and thus identifying novel potential targets in such retinal diseases., (Copyright © 2023 The Authors. Published by Elsevier GmbH.. All rights reserved.)

The hexosamine biosynthetic pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc) to facilitate O-linked GlcNAc (O-GlcNAc) protein modifications, and subsequently enhance cell survival under lethal stresses. Transcript induced in spermiogenesis 40 (Tisp40) is an endoplasmic reticulum membrane-resident transcription factor and plays critical roles in cell homeostasis. Here, we show that Tisp40 expression, cleavage and nuclear accumulation are increased by cardiac ischemia/reperfusion (I/R) injury. Global Tisp40 deficiency exacerbates, whereas cardiomyocyte-restricted Tisp40 overexpression ameliorates I/R-induced oxidative stress, apoptosis and acute cardiac injury, and modulates cardiac remodeling and dysfunction following long-term observations in male mice. In addition, overexpression of nuclear Tisp40 is sufficient to attenuate cardiac I/R injury in vivo and in vitro. Mechanistic studies indicate that Tisp40 directly binds to a conserved unfolded protein response element (UPRE) of the glutamine-fructose-6-phosphate transaminase 1 (GFPT1) promoter, and subsequently potentiates HBP flux and O-GlcNAc protein modifications. Moreover, we find that I/R-induced upregulation, cleavage and nuclear accumulation of Tisp40 in the heart are mediated by endoplasmic reticulum stress. Our findings identify Tisp40 as a cardiomyocyte-enriched UPR-associated transcription factor, and targeting Tisp40 may develop effective approaches to mitigate cardiac I/R injury., (© 2023. The Author(s).)

Objective: A buildup of skeletal muscle plasma membrane (PM) cholesterol content in mice occurs within 1 week of a Western-style high-fat diet and causes insulin resistance. The mechanism driving this cholesterol accumulation and insulin resistance is not known. Promising cell data implicate that the hexosamine biosynthesis pathway (HBP) triggers a cholesterolgenic response via increasing the transcriptional activity of Sp1. In this study we aimed to determine whether increased HBP/Sp1 activity represented a preventable cause of insulin resistance., Methods: C57BL/6NJ mice were fed either a low-fat (LF, 10% kcal) or high-fat (HF, 45% kcal) diet for 1 week. During this 1-week diet the mice were treated daily with either saline or mithramycin-A (MTM), a specific Sp1/DNA-binding inhibitor. A series of metabolic and tissue analyses were then performed on these mice, as well as on mice with targeted skeletal muscle overexpression of the rate-limiting HBP enzyme glutamine-fructose-6-phosphate-amidotransferase (GFAT) that were maintained on a regular chow diet., Results: Saline-treated mice fed this HF diet for 1 week did not have an increase in adiposity, lean mass, or body mass while displaying early insulin resistance. Consistent with an HBP/Sp1 cholesterolgenic response, Sp1 displayed increased O-GlcNAcylation and binding to the HMGCR promoter that increased HMGCR expression in skeletal muscle from saline-treated HF-fed mice. Skeletal muscle from these saline-treated HF-fed mice also showed a resultant elevation of PM cholesterol with an accompanying loss of cortical filamentous actin (F-actin) that is essential for insulin-stimulated glucose transport. Treating these mice daily with MTM during the 1-week HF diet fully prevented the diet-induced Sp1 cholesterolgenic response, loss of cortical F-actin, and development of insulin resistance. Similarly, increases in HMGCR expression and cholesterol were measured in muscle from GFAT transgenic mice compared to age- and weight-match wildtype littermate control mice. In the GFAT Tg mice we found that these increases were alleviated by MTM., Conclusions: These data identify increased HBP/Sp1 activity as an early mechanism of diet-induced insulin resistance. Therapies targeting this mechanism may decelerate T2D development., (Copyright © 2023 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate- N -acetyl glucosamine, UDP-GlcNAc, which is a key metabolite that is used for N - or O -linked glycosylation, a co- or post-translational modification, respectively, that modulates protein activity and expression. The production of hexosamines can occur via de novo or salvage mechanisms that are catalyzed by metabolic enzymes. Nutrients including glutamine, glucose, acetyl-CoA, and UTP are utilized by the HBP. Together with availability of these nutrients, signaling molecules that respond to environmental signals, such as mTOR, AMPK, and stress-regulated transcription factors, modulate the HBP. This review discusses the regulation of GFAT, the key enzyme of the de novo HBP, as well as other metabolic enzymes that catalyze the reactions to produce UDP-GlcNAc. We also examine the contribution of the salvage mechanisms in the HBP and how dietary supplementation of the salvage metabolites glucosamine and N -acetylglucosamine could reprogram metabolism and have therapeutic potential. We elaborate on how UDP-GlcNAc is utilized for N -glycosylation of membrane and secretory proteins and how the HBP is reprogrammed during nutrient fluctuations to maintain proteostasis. We also consider how O -GlcNAcylation is coupled to nutrient availability and how this modification modulates cell signaling. We summarize how deregulation of protein N -glycosylation and O -GlcNAcylation can lead to diseases including cancer, diabetes, immunodeficiencies, and congenital disorders of glycosylation. We review the current pharmacological strategies to inhibit GFAT and other enzymes involved in the HBP or glycosylation and how engineered prodrugs could have better therapeutic efficacy for the treatment of diseases related to HBP deregulation.

Osteoclasts play a key role in the regulation of bone mass and are highly active metabolically. Here we show that a metabolic reprogramming toward the hexosamine biosynthetic pathway (HBP) is required not only for osteoclast differentiation but also to determine the bone resorption mode during physiological and pathological bone remodeling. We found that pharmacological inhibition of O-GlcNAc transferase (OGT) significantly reduced protein O-GlcNAcylation and osteoclast differentiation. Accordingly, genetic deletion of OGT also inhibited osteoclast formation and downregulated critical markers related to osteoclasts differentiation and function (NFATc1, α v integrin, cathepsin K). Indeed, cells treated with OSMI-1, an OGT inhibitor, also reduced nuclear translocation of NFATc1. Furthermore, the addition of exogenous N-acetylglucosamine (GlcNAc) strongly increased osteoclast formation and demineralization ability. Strikingly, our data show for the first time that O-GlcNAcylation facilitates an aggressive trench resorption mode in human cells. The incubation of osteoclasts with exogenous GlcNAc increases the percentage of erosion by trench while having no effect on pit resorption mode. Through time-lapse recording, we documented that osteoclasts making trenches moving across the bone surface are sensitive to GlcNAcylation. Finally, osteoclast-specific Ogt -deficient mice show increased bone density and reduced inflammation-induced bone loss during apical periodontitis model. We show that osteoclast-specific Og t-deficient mice are less susceptible to develop bacterial-induced periapical lesion. Consistent with this, Ogt -deleted mice showed a decreased number of tartrate-resistant acid phosphatase-positive cells lining the apical periodontitis site. In summary, here we describe a hitherto undiscovered role of the HBP/O-GlcNAcylation axis tuning resorption mode and dictating bone resorption outcome.

Altered cellular metabolism is a hallmark of cancer pathogenesis and progression; for example, a near-universal feature of cancer is increased metabolic flux through the hexosamine biosynthetic pathway (HBP). This pathway produces uridine diphosphate N -acetylglucosamine (UDP-GlcNAc), a potent oncometabolite that drives multiple facets of cancer progression. In this study, we synthesized and evaluated peracetylated hexosamine analogs designed to reduce flux through the HBP. By screening a panel of analogs in pancreatic cancer and glioblastoma multiform (GBM) cells, we identified Ac 4 Glc2Bz─a benzyl-modified GlcNAc mimetic─as an antiproliferative cancer drug candidate that down-regulated oncogenic metabolites and reduced GBM cell motility at concentrations non-toxic to non-neoplastic cells. More specifically, the growth inhibitory effects of Ac 4 Glc2Bz were linked to reduced levels of UDP-GlcNAc and concomitant decreases in protein O-GlcNAc modification in both pancreatic cancer and GBM cells. Targeted metabolomics analysis in GBM cells showed that Ac 4 Glc2Bz disturbed glucose metabolism, amino acid pools, and nucleotide precursor biosynthesis, consistent with reduced proliferation and other anti-oncogenic properties of this analog. Furthermore, Ac 4 Glc2Bz reduced the invasion, migration, and stemness of GBM cells. Importantly, normal metabolic functions mediated by UDP-GlcNAc were not disrupted in non-neoplastic cells, including maintenance of endogenous levels of O-GlcNAcylation with no global disruption of N-glycan production. Finally, a pilot in vivo study showed that a potential therapeutic window exists where animals tolerated 5- to 10-fold higher levels of Ac 4 Glc2Bz than projected for in vivo efficacy. Together, these results establish GlcNAc analogs targeting the HBP through salvage mechanisms as a new therapeutic approach to safely normalize an important facet of aberrant glucose metabolism associated with cancer.

Metabolite sensing, a fundamental biological process, plays a key role in metabolic signaling circuit rewiring. Hexosamine biosynthetic pathway (HBP) is a glucose metabolic pathway essential for the synthesis of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which senses key nutrients and integrally maintains cellular homeostasis. UDP-GlcNAc dynamically regulates protein N-glycosylation and O-linked-N-acetylglucosamine modification (O-GlcNAcylation). Dysregulated HBP flux leads to abnormal protein glycosylation, and contributes to cancer development and progression by affecting protein function and cellular signaling. Furthermore, O-GlcNAcylation regulates cellular signaling pathways, and its alteration is linked to various cancer characteristics. Additionally, recent findings have suggested a close association between HBP stimulation and cancer stemness; an elevated HBP flux promotes cancer cell conversion to cancer stem cells and enhances chemotherapy resistance via downstream signal activation. In this review, we highlight the prominent roles of HBP in metabolic signaling and summarize the recent advances in HBP and its downstream signaling, relevant to cancer., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Natural killer T (NKT) cells operate distinctly different metabolic programming from CD4 T cells, including a strict requirement for glutamine to regulate cell homeostasis. However, the underlying mechanisms remain unknown. Here, we report that at a steady state, NKT cells have higher glutamine levels than CD4 T cells and that NKT cells increase glutaminolysis on activation. Activated NKT cells use glutamine to fuel the tricarboxylic acid cycle and glutathione synthesis. In addition, glutamine-derived nitrogen enables protein glycosylation via the hexosamine biosynthesis pathway (HBP). Each of these branches of glutamine metabolism seems to be critical for NKT cell homeostasis and mitochondrial functions. Glutaminolysis and HBP differentially regulate interleukin-4 (IL-4) and interferon γ (IFNγ) production. Glutamine metabolism appears to be controlled by AMP-activated protein kinase (AMPK)-mammalian target of rapamycin complex 1 (mTORC1) signaling. These findings highlight a distinct metabolic requirement of NKT cells compared with CD4 T cells, which may have therapeutic implications in the treatment of certain nutrient-restricted diseases., Competing Interests: Declaration of interests C.A.L. has received consulting fees from Astellas Pharmaceuticals and Odyssey Therapeutics and is an inventor on patents pertaining to K-Ras-regulated metabolic pathways and redox-control pathways in cancer and targeting the glutamic-oxaloacetic transaminase 1 (GOT1) pathway as a therapeutic approach., (Copyright © 2022 The Authors. Published by Elsevier Inc. All rights reserved.)

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Cairns, Joseph and Essop.)

Establishing an ideal human follicle culture system for oncofertility patients relies mainly on animal models since donor tissue is scarce and often of suboptimal quality. The in vitro system developed in our laboratory supports the growth of prepubertal mouse secondary follicles up to mature oocytes. Given the importance of glucose in preparing the oocyte for proper maturation, a baseline characterization of follicle metabolism both in the culture system and in vivo was carried out. Markers of glucose-related pathways (glycolysis, tricarboxylic acid [TCA] cycle, pentose phosphate pathway [PPP], polyol pathway, and hexosamine biosynthetic pathway), as well as the antioxidant capacity, were measured in the different follicle cell types by both enzymatic activities (spectrophotometric detection) and gene expression (qPCR). This study confirmed that in vivo the somatic cells, mainly granulosa, exhibit intense glycolytic activity, while oocytes perform PPP. Throughout the final maturation step, oocytes in vivo and in vitro showed steady levels for all the key enzymes and metabolites. On the other hand, ovulation triggers a boost of pyruvate and lactate uptake in cumulus cells in vivo, consumes reduced nicotinamide adenine dinucleotide phosphate, and increases TCA cycle and small molecules antioxidant capacity activities, while in vitro, the metabolic upregulation in all the studied pathways is limited. This altered metabolic pattern might be a consequence of cell exhaustion because of culture conditions, impeding cumulus cells to fulfill their role in providing proper support for acquiring oocyte competence., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for the Study of Reproduction. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

A malformed tumour vascular network provokes the nutrient-deprived tumour microenvironment (TME), which conversely activates endothelial cell (EC) functions and stimulates neovascularization. Emerging evidence suggests that the flexible metabolic adaptability of tumour cells helps to establish a metabolic symbiosis among various cell subpopulations in the fluctuating TME. In this study, the authors propose a novel metabolic link between bladder cancer (BCa) cells and ECs in the nutrient-scarce TME, in which BCa-secreted glutamine-fructose-6-phosphate aminotransferase 1 (GFAT1) via small extracellular vesicles (sEVs) reprograms glucose metabolism by increasing hexosamine biosynthesis pathway flux in ECs and thus enhances O-GlcNAcylation. Moreover, seryl-tRNA synthetase (SerRS) O-GlcNAcylation at serine 101 in ECs promotes its degradation by ubiquitination and impeded importin α5-mediated nuclear translocation. Intranuclear SerRS attenuates vascular endothelial growth factor transcription by competitively binding to the GC-rich region of the proximal promotor. Additionally, GFAT1 knockout in tumour cells blocks SerRS O-GlcNAcylation in ECs and attenuates angiogenesis both in vitro and in vivo. However, administration of GFAT1-overexpressing BCa cells-derived sEVs increase the angiogenetic activity in the ECs of GFAT1-knockout mice. In summary, this study suggests that inhibiting sEV-mediated GFAT1 secretion from BCa cells and targeting SerRS O-GlcNAcylation in ECs may serve as novel strategies for BCa antiangiogenetic therapy., (© 2022 The Authors. Advanced Science published by Wiley-VCH GmbH.)

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., (© 2022 The Authors. Aging Cell published by Anatomical Society and John Wiley & Sons Ltd.)

Kidneys maintain internal milieu homeostasis through a well-regulated manipulation of body fluid composition. This task is performed by the correlation between structure and function in the nephron. Kidney diseases are chronic conditions impacting healthcare programs globally, and despite efforts, therapeutic options for its treatment are limited. The development of chronic degenerative diseases is associated with changes in protein O-GlcNAcylation, a post-translation modification involved in the regulation of diverse cell function. O-GlcNAcylation is regulated by the enzymatic balance between O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA) which add and remove GlcNAc residues on target proteins, respectively. Furthermore, the hexosamine biosynthetic pathway provides the substrate for protein O-GlcNAcylation. Beyond its physiological role, several reports indicate the participation of protein O-GlcNAcylation in cardiovascular, neurodegenerative, and metabolic diseases. In this review, we discuss the impact of protein O-GlcNAcylation on physiological renal function, disease conditions, and possible future directions in the field.

The cell wall (CW) of fungi exhibits a complex structure and a characteristic chemical composition consisting almost entirely of interacting crystalline and amorphous polysaccharides. These are synthesized by a number of sugar polymerases and depolymerases encoded by a high proportion of the fungal genome (for instance, 20% in Saccharomyces cerevisiae ). These enzymes act in an exquisitely coordinated process to assemble the tridimensional and the functional structure of the wall. Apart from playing a critical role in morphogenesis, cell protection, viability and pathogenesis, the CW represents a potential target for antifungals as most of its constituents do not exist in humans. Chitin, β-glucans and cellulose are the most frequent crystalline polymers found in the fungal CW. The hexosamine biosynthesis pathway (HBP) is critical for CW elaboration. Also known as the Leloir pathway, this pathway ends with the formation of UDP-N-GlcNAc after four enzymatic steps that start with fructose-6-phosphate and L-glutamine in a short deviation of glycolysis. This activated aminosugar is used for the synthesis of a large variety of biomacromolecules in a vast number of organisms including bacteria, fungi, insects, crustaceans and mammalian cells. The first reaction of the HBP is catalyzed by GlcN-6-P synthase (L-glutamine:D-fructose-6-phosphate amidotransferase; EC 2.6.1.16), a critical enzyme that has been considered as a potential target for antifungals. The enzyme regulates the amount of cell UDP-N-GlcNAc and in eukaryotes is feedback inhibited by the activated aminosugar and other factors. The native and recombinant forms of GlcN-6-P synthase has been purified and characterized from both prokaryotic and eukaryotic organisms and demonstrated its critical role in CW remodeling and morphogenesis after exposure of some fungi to agents that stress the cell surface by interacting with wall polymers. This review deals with some of the cell compensatory responses of fungi to wall damage induced by Congo Red and Calcofluor White., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Ortiz-Ramírez, Cuéllar-Cruz and López-Romero.)

The hexosamine biosynthetic pathway (HBP) is connected to abnormal N- and O-linked protein glycosylation in cancer, which performs critical roles in tumorigenesis. However, the regulation mechanisms of HBP and its role in colorectal cancer (CRC) progression remain unexplained. This study analyzed the expression level of phosphoglucomutase 3 (PGM3), a key enzyme in HBP, and identified its function in CRC cell lines. Analysis of publicly available CRC microarray data determined that PGM3 is upregulated in CRC tumor tissues. Furthermore, functional experiments emphasized the significant roles of PGM3 in facilitating CRC cell proliferation and migration. Mechanistically, we demonstrated that the activity of β-catenin in CRC was maintained by PGM3-mediated O-GlcNAcylation. PGM3 knockdown or inhibition of O-GlcNAc transferase decreased β-catenin activity and the expression levels of its downstream targets. Collectively, our findings indicate that PGM3 exhibits tumor-promoting roles by elevating O-GlcNAcylation level and maintaining β-catenin activity, and might serve as a prognostic biomarker and treatment target in CRC.

Respiratory syncytial virus (RSV), or human orthopneumovirus, is a negative-sense RNA virus that is the causative agent of severe lower respiratory tract infections in children and is associated with exacerbations of adult lung disease. The mechanisms how severe and/or repetitive virus infections cause declines in pulmonary capacity are not fully understood. We have recently discovered that viral replication triggers epithelial plasticity and metabolic reprogramming involving the hexosamine biosynthetic pathway (HBP). In this study, we examine the relationship between viral induced innate inflammation and the activation of hexosamine biosynthesis in small airway epithelial cells. We observe that RSV induces ~2-fold accumulation of intracellular UDP-GlcNAc, the end-product of the HBP and the obligate substrate of N glycosylation. Using two different silencing approaches, we observe that RSV replication activates the HBP pathway in a manner dependent on the RELA proto-oncogene (65 kDa subunit). To better understand the effect of RSV on the cellular N glycoproteome, and its RELA dependence, we conduct affinity enriched LC-MS profiling in wild-type and RELA-silenced cells. We find that RSV induces the accumulation of 171 N glycosylated peptides in a RELA-dependent manner; these proteins are functionally enriched in integrins and basal lamina formation. To elaborate this mechanism of HBP expression, we demonstrate that RSV infection coordinately induces the HBP pathway enzymes in a manner requiring RELA; these genes include Glutamine-Fructose-6-Phosphate Transaminase 1 (GFPT)-1/2, Glucosamine-Phosphate N-Acetyltransferase (GNPNAT)-1, phosphoglucomutase (PGM)-3 and UDP-N-Acetylglucosamine Pyrophosphorylase (UAP)-1. Using small-molecule inhibitor(s) of 8-oxoguanine DNA glycosylase1 (OGG1), we observe that OGG1 is also required for the expression of HBP pathway. In proximity ligation assays, RSV induces the formation of a nuclear and mitochondrial RELA∙OGG1 complex. In co-immunoprecipitaton (IP) experiments, we discover that RSV induces Ser 536-phosphorylated RELA to complex with OGG1. Chromatin IP experiments demonstrate a major role of OGG1 in supporting the recruitment of RELA and phosphorylated RNA Pol II to the HBP pathway genes. We conclude that the RELA∙OGG1 complex is an epigenetic regulator mediating metabolic reprogramming and N glycoprotein modifications of integrins in response to RSV. These findings have implications for viral-induced adaptive epithelial responses.

The glucose-requiring hexosamine biosynthetic pathway (HBP), which produces UDP-N-acetylglucosamine for glycosylation reactions, promotes lung adenocarcinoma (LUAD) progression. However, lung tumor cells often reside in low-nutrient microenvironments, and whether the HBP is involved in the adaptation of LUAD to nutrient stress is unknown. Here, we show that the HBP and the coat complex II (COPII) play a key role in cell survival during glucose shortage. HBP up-regulation withstood low glucose-induced production of proteins bearing truncated N -glycans, in the endoplasmic reticulum. This function for the HBP, alongside COPII up-regulation, rescued cell surface expression of a subset of glycoproteins. Those included the epidermal growth factor receptor (EGFR), allowing an EGFR-dependent cell survival under low glucose in anchorage-independent growth. Accordingly, high expression of the HBP rate-limiting enzyme GFAT1 was associated with wild-type EGFR activation in LUAD patient samples. Notably, HBP and COPII up-regulation distinguished LUAD from the lung squamous-cell carcinoma subtype, thus uncovering adaptive mechanisms of LUAD to their harsh microenvironment., (© 2022 Dragic et al.)

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., Competing Interests: VK, SR, MH, KA, LE, SB, SM, UE, BS, UB, MD No competing interests declared, (© 2022, Kroef et al.)

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

It is well established that diabetes and its associated hyperglycemia negatively impact retinal function, yet we know little about the role played by augmented flux through the Hexosamine Biosynthetic Pathway (HBP). This offshoot of the glycolytic pathway produces UDP-Nacetyl- glucosamine, which serves as the substrate for post-translational O-linked modification of proteins in a process referred to as O-GlcNAcylation. HBP flux and subsequent protein O-GlcNAcylation serve as nutrient sensors, enabling cells to integrate metabolic information to appropriately modulate fundamental cellular processes including gene expression. Here we summarize the impact of diabetes on retinal physiology, highlighting recent studies that explore the role of O-GlcNAcylation- induced variation in mRNA translation in retinal dysfunction and the pathogenesis of Diabetic Retinopathy (DR). Augmented O-GlcNAcylation results in wide variation in the selection of mRNAs for translation, in part, due to O-GlcNAcylation of the translational repressor 4E-BP1. Recent studies demonstrate that 4E-BP1 plays a critical role in regulating O-GlcNAcylation-induced changes in the translation of the mRNAs encoding Vascular Endothelial Growth Factor (VEGF), a number of important mitochondrial proteins, and CD40, a key costimulatory molecule involved in diabetes-induced retinal inflammation. Remarkably, 4E-BP1/2 ablation delays the onset of diabetes- induced visual dysfunction in mice. Thus, pharmacological interventions to prevent the impact of O-GlcNAcylation on 4E-BP1 may represent promising therapeutics to address the development and progression of DR. In this regard, we discuss the potential interplay between retinal O-GlcNAcylation and the ocular renin-angiotensin system as a potential therapeutic target of future interventions., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

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., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

Tumors frequently exhibit aberrant glycosylation, which can impact cancer progression and therapeutic responses. The hexosamine biosynthesis pathway (HBP) produces uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a major substrate for glycosylation in the cell. Prior studies have identified the HBP as a promising therapeutic target in pancreatic ductal adenocarcinoma (PDA). The HBP requires both glucose and glutamine for its initiation. The PDA tumor microenvironment is nutrient poor, however, prompting us to investigate how nutrient limitation impacts hexosamine synthesis. Here, we identify that glutamine limitation in PDA cells suppresses de novo hexosamine synthesis but results in increased free GlcNAc abundance. GlcNAc salvage via N-acetylglucosamine kinase (NAGK) is engaged to feed UDP-GlcNAc pools. NAGK expression is elevated in human PDA, and NAGK deletion from PDA cells impairs tumor growth in mice. Together, these data identify an important role for NAGK-dependent hexosamine salvage in supporting PDA tumor growth., Competing Interests: SC, CM, LI, HA, MN, BS, TT, KS, ST, SK, JB, KW No competing interests declared, IB IAB is a founder of Proteoform Bio and a paid consultant for Calico, Chimerix, PTC Therapeutics, Takeda Pharmaceuticals, and Vivo Capital., (© 2021, Campbell et al.)

Women with pre-existing diabetes have an increased risk of poor pregnancy outcomes, including disordered fetal growth, caused by changes to placental function. Here we investigate the possibility that the hexosamine biosynthetic pathway, which utilises cellular nutrients to regulate protein function via post-translationally modification with O-linked N-acetylglucosamine (GlcNAc), mediates the placental response to the maternal metabolic milieu. Mass spectrometry analysis revealed that the placental O-GlcNAcome is altered in women with type 1 (n = 6) or type 2 (n = 6) diabetes T2D (≥ twofold change in abundance in 162 and 165 GlcNAcylated proteins respectively compared to BMI-matched controls n = 11). Ingenuity pathway analysis indicated changes to clathrin-mediated endocytosis (CME) and CME-associated proteins, clathrin, Transferrin (TF), TF receptor and multiple Rabs, were identified as O-GlcNAcylation targets. Stimulating protein O-GlcNAcylation using glucosamine (2.5 mM) increased the rate of TF endocytosis by human placental cells (p = 0.02) and explants (p = 0.04). Differential GlcNAcylation of CME proteins suggests altered transfer of cargo by placentas of women with pre-gestational diabetes, which may contribute to alterations in fetal growth. The human placental O-GlcNAcome provides a resource to aid further investigation of molecular mechanisms governing placental nutrient sensing., (© 2021. The Author(s).)

There are five known general catalytic mechanisms used by enzymes to catalyze carbohydrate epimerization. The amino sugar epimerase N-acetylmannosamine-6-phosphate 2-epimerase (NanE) has been proposed to use a deprotonation-reprotonation mechanism, with an essential catalytic lysine required for both steps. However, the structural determinants of this mechanism are not clearly established. We characterized NanE from Staphylococcus aureus using a new coupled assay to monitor NanE catalysis in real time and found that it has kinetic constants comparable with other species. The crystal structure of NanE from Staphylococcus aureus, which comprises a triosephosphate isomerase barrel fold with an unusual dimeric architecture, was solved with both natural and modified substrates. Using these substrate-bound structures, we identified the following active-site residues lining the cleft at the C-terminal end of the β-strands: Gln11, Arg40, Lys63, Asp124, Glu180, and Arg208, which were individually substituted and assessed in relation to the mechanism. From this, we re-evaluated the central role of Glu180 in this mechanism alongside the catalytic lysine. We observed that the substrate is bound in a conformation that ideally positions the C5 hydroxyl group to be activated by Glu180 and donate a proton to the C2 carbon. Taken together, we propose that NanE uses a novel substrate-assisted proton displacement mechanism to invert the C2 stereocenter of N-acetylmannosamine-6-phosphate. Our data and mechanistic interpretation may be useful in the development of inhibitors of this enzyme or in enzyme engineering to produce biocatalysts capable of changing the stereochemistry of molecules that are not amenable to synthetic methods., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2021 The Authors. Published by Elsevier Inc. All rights reserved.)

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.

Cancer cells generally exhibit higher metabolic demands relative to that of normal tissue cells. This offers great possibilities to exploit metabolic glycoengineering in combination with bio-orthogonal chemistry reactions to achieve tumour site-targeted therapeutic delivery. This work addresses the selectivity of metabolic glycan labelling in diseased (i.e., cancer) versus normal cells grown in a multicellular environment. Dibenzocylooctyne (DBCO)-bearing acetylated-d-mannosamine (Ac4ManNDBCO) was synthesised to metabolically label three different types of cell lines originating from the human lung tissues: A549 adenocarcinomic alveolar basal epithelial cells, MeT5A non-cancerous mesothelial cells, and MRC5 non-cancerous fibroblasts. These cell lines displayed different labelling sensitivity, which trended with their doubling time in the following order: A549 ≈ MeT5A > MRC5. The higher metabolic labelling efficiency inherently led to a higher extent of specific binding and accumulation of the clickable N3-conjugated gold nanoparticles (N3-AuNps, core diameter = 30 nm) in the DBCO-glycan modified A549 and MeT5A cells, but to a less prominent effect in MRC5 cells. These findings demonstrate that relative rates of cell metabolism can be exploited using metabolic labelling to recruit nanotherapeutics whilst minimising non-specific targeting of surrounding tissues.

The hexosamine biosynthetic pathway (HBP) is essential for the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the building block of glycosaminoglycans, thus playing a crucial role in cartilage anabolism. Although O-GlcNAcylation represents a protective regulatory mechanism in cellular processes, it has been associated with degenerative diseases, including osteoarthritis (OA). The present study focuses on HBP-related processes as potential therapeutic targets after cartilage trauma. Human cartilage explants were traumatized and treated with GlcNAc or glucosamine sulfate (GS); PUGNAc, an inhibitor of O-GlcNAcase; or azaserine (AZA), an inhibitor of GFAT-1. After 7 days, cell viability and gene expression analysis of anabolic and catabolic markers, as well as HBP-related enzymes, were performed. Moreover, expression of catabolic enzymes and type II collagen (COL2) biosynthesis were determined. Proteoglycan content was assessed after 14 days. Cartilage trauma led to a dysbalanced expression of different HBP-related enzymes, comparable to the situation in highly degenerated tissue. While GlcNAc and PUGNAc resulted in significant cell protection after trauma, only PUGNAc increased COL2 biosynthesis. Moreover, PUGNAc and both glucosamine derivatives had anti-catabolic effects. In contrast, AZA increased catabolic processes. Overall, "fueling" the HBP by means of glucosamine derivatives or inhibition of deglycosylation turned out as cells and chondroprotectives after cartilage trauma.

The hexosamine pathway (HP) is a key anabolic pathway whose product uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc) is an essential precursor for glycosylation processes in mammals. It modulates the ER stress response and HP activation extends lifespan in Caenorhabditis elegans. The highly conserved glutamine fructose-6-phosphate amidotransferase 1 (GFAT-1) is the rate-limiting HP enzyme. GFAT-1 activity is modulated by UDP-GlcNAc feedback inhibition and via phosphorylation by protein kinase A (PKA). Molecular consequences of GFAT-1 phosphorylation, however, remain poorly understood. Here, we identify the GFAT-1 R203H substitution that elevates UDP-GlcNAc levels in C. elegans. In human GFAT-1, the R203H substitution interferes with UDP-GlcNAc inhibition and with PKA-mediated Ser205 phosphorylation. Our data indicate that phosphorylation affects the interactions of the two GFAT-1 domains to control catalytic activity. Notably, Ser205 phosphorylation has two discernible effects: it lowers baseline GFAT-1 activity and abolishes UDP-GlcNAc feedback inhibition. PKA controls the HP by uncoupling the metabolic feedback loop of GFAT-1.

Cancer cells characteristically have a high proliferation rate. Because tumor growth depends on energy-consuming anabolic processes, including biosynthesis of protein, lipid, and nucleotides, many tumor-associated conditions, including intermittent oxygen deficiency due to insufficient vascularization, oxidative stress, and nutrient deprivation, results from fast growth. To cope with these environmental stressors, cancer cells, including cancer stem cells, must adapt their metabolism to maintain cellular homeostasis. It is well- known that cancer stem cells (CSC) reprogram their metabolism to adapt to live in hypoxic niches. They usually change from oxidative phosphorylation to increased aerobic glycolysis even in the presence of oxygen. However, as opposed to most differentiated cancer cells relying on glycolysis, CSCs can be highly glycolytic or oxidative phosphorylation-dependent, displaying high metabolic plasticity. Although the influence of the metabolic and nutrient-sensing pathways on the maintenance of stemness has been recognized, the molecular mechanisms that link these pathways to stemness are not well known. Here in this review, we describe the most relevant signaling pathways involved in nutrient sensing and cancer cell survival. Among them, Adenosine monophosphate (AMP)-activated protein kinase (AMPK) pathway, mTOR pathway, and Hexosamine Biosynthetic Pathway (HBP) are critical sensors of cellular energy and nutrient status in cancer cells and interact in complex and dynamic ways., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Robles-Flores, Moreno-Londoño and Castañeda-Patlán.)

Metabolic glycoengineering enables a directed modification of cell surfaces by introducing target molecules to surface proteins displaying new features. Biochemical pathways involving glycans differ in dependence on the cell type; therefore, this technique should be tailored for the best results. We characterized metabolic glycoengineering in telomerase-immortalized human mesenchymal stromal cells (hMSC-TERT) as a model for primary hMSC, to investigate its applicability in TERT-modified cell lines. The metabolic incorporation of N -azidoacetylmannosamine (Ac 4 ManNAz) and N -alkyneacetylmannosamine (Ac 4 ManNAl) into the glycocalyx as a first step in the glycoengineering process revealed no adverse effects on cell viability or gene expression, and the in vitro multipotency (osteogenic and adipogenic differentiation potential) was maintained under these adapted culture conditions. In the second step, glycoengineered cells were modified with fluorescent dyes using Cu-mediated click chemistry. In these analyses, the two mannose derivatives showed superior incorporation efficiencies compared to glucose and galactose isomers. In time-dependent experiments, the incorporation of Ac 4 ManNAz was detectable for up to six days while Ac 4 ManNAl-derived metabolites were absent after two days. Taken together, these findings demonstrate the successful metabolic glycoengineering of immortalized hMSC resulting in transient cell surface modifications, and thus present a useful model to address different scientific questions regarding glycosylation processes in skeletal precursors.

This report describes novel thiol-modified N -acetylmannosamine (ManNAc) analogs that extend metabolic glycoengineering (MGE) applications of Ac 5 ManNTGc, a non-natural monosaccharide that metabolically installs the thio-glycolyl of sialic acid into human glycoconjugates. We previously found that Ac 5 ManNTGc elicited non-canonical activation of Wnt signaling in human embryoid body derived (hEBD) cells but only in the presence of a high affinity, chemically compatible scaffold. Our new analogs Ac 5 ManNTProp and Ac 5 ManNTBut overcome the requirement for a complementary scaffold by displaying thiol groups on longer, N -acyl linker arms, thereby presumably increasing their ability to interact and crosslink with surrounding thiols. These new analogs showed increased potency in human neural stem cells (hNSCs) and human adipose stem cells (hASCs). In the hNSCs, Ac 5 ManNTProp upregulated biochemical endpoints consistent with Wnt signaling in the absence of a thiol-reactive scaffold. In the hASCs, both Ac 5 ManNTProp and Ac 5 ManNTBut suppressed adipogenic differentiation, with Ac 5 ManNTBut providing a more potent response, and they did not interfere with differentiation to a glial lineage (Schwann cells). These results expand the horizon for using MGE in regenerative medicine by providing new tools (Ac 5 ManNTProp and Ac 5 ManNTBut) for manipulating human stem cells.

Oxidative stress (OS) is defined as a disturbance in the prooxidant-antioxidant balance of the cell, in favor of the former, which results in the antioxidant capacity of the cell to be overpowered. Excess reactive oxygen species (ROS) production is very harmful to cell constituents, especially proteins, lipids, and DNA, thus causing damage to the cell. Oxidative stress has been associated with a variety of pathologic conditions, including diabetes mellitus (DM), cancer, atherosclerosis, neurodegenerative diseases, rheumatoid arthritis, ischemia/reperfusion injury, obstructive sleep apnea, and accelerated aging. Regarding DM specifically, previous experimental and clinical studies have pointed to the fact that oxidative stress probably plays a major role in the pathogenesis and development of diabetic complications. It is postulated that hyperglycemia induces free radicals and impairs endogenous antioxidant defense systems through several different mechanisms. In particular, hyperglycemia promotes the creation of advanced glycation end-products (AGEs), the activation of protein kinase C (PKC), and the hyperactivity of hexosamine and sorbitol pathways, leading to the development of insulin resistance, impaired insulin secretion, and endothelial dysfunction, by inducing excessive ROS production and OS. Furthermore, glucose variability has been associated with OS as well, and recent evidence suggests that also hypoglycemia may be playing an important role in favoring diabetic vascular complications through OS, inflammation, prothrombotic events, and endothelial dysfunction. The association of these diabetic parameters (i.e., hyperglycemia, glucose variability, and hypoglycemia) with oxidative stress will be reviewed here., Competing Interests: The authors declare no conflict of interest., (Copyright © 2020 Eleftheria Papachristoforou et al.)

The hexosamine biosynthetic pathway (HBP) converts glucose to uridine-diphosphate- N -acetylglucosamine, which, when added to serines or threonines, modulates protein function through protein O-GlcNAcylation. Glutamine-fructose-6-phosphate amidotransferase (GFAT) regulates HBP flux, and AMP-kinase phosphorylation of GFAT blunts GFAT activity and O-GlcNAcylation. While numerous studies demonstrate increased right ventricle (RV) glucose uptake in pulmonary arterial hypertension (PAH), the relationship between O-GlcNAcylation and RV function in PAH is unexplored. Therefore, we examined how colchicine-mediated AMP-kinase activation altered HBP intermediates, O-GlcNAcylation, mitochondrial function, and RV function in pulmonary artery-banded (PAB) and monocrotaline (MCT) rats. AMPK activation induced GFAT phosphorylation and reduced HBP intermediates and O-GlcNAcylation in MCT but not PAB rats. Reduced O-GlcNAcylation partially restored the RV metabolic signature and improved RV function in MCT rats. Proteomics revealed elevated expression of O-GlcNAcylated mitochondrial proteins in MCT RVs, which fractionation studies corroborated. Seahorse micropolarimetry analysis of H9c2 cardiomyocytes demonstrated colchicine improved mitochondrial function and reduced O-GlcNAcylation. Presence of diabetes in PAH, a condition of excess O-GlcNAcylation, reduced RV contractility when compared to nondiabetics. Furthermore, there was an inverse relationship between RV contractility and HgbA1C. Finally, RV biopsy specimens from PAH patients displayed increased O-GlcNAcylation. Thus, excess O-GlcNAcylation may contribute to metabolic derangements and RV dysfunction in PAH.

Understanding how a cell membrane protein functions on living cells remains a challenge for cell biology. Specific placement of functional molecules on specific proteins in their native environment would allow comprehensive study of proteins' dynamic functions. Existing methods cannot facilely achieve multiple modifications on specific membrane proteins. In this report, we describe an aptamer-induced, protein-specific bio-orthogonal modification technology for precise nongenetic immobilization of multiple small functional molecules on target membrane glycoproteins by combining metabolic technology and aptamer targeting. In brief, DNA probes were designed by modifying aptamers, which bind to target proteins on the surfaces of living cells pretreated with N -azidoacetylmannosamine-tetraacylated (Ac 4 ManNAz). The cyclooctynes tagged of DNA probes will approach the azide groups to trigger the bio-orthogonal reactions. After UV irradiation and hybridization with cDNA (complementary DNA), the aptamers can be removed, and the process can be repeated to achieve multiple modifications for multicolor imaging and cell surface nanoengineering on specific proteins.