Hexosamine Biosynthesis Pathway Research Articles

Revealing prognostic insights of programmed cell death (PCD)-associated genes in advanced non-small cell lung cancer.

The management of patients with advanced non-small cell lung cancer (NSCLC) presents significant challenges due to cancer cells' intricate and heterogeneous nature. Programmed cell death (PCD) pathways are crucial in diverse biological processes. Nevertheless, the prognostic significance of cell death in NSCLC remains incompletely understood. Our study aims to investigate the prognostic importance of PCD genes and their ability to precisely stratify and evaluate the survival outcomes of patients with advanced NSCLC. We employed Weighted Gene Co-expression Network Analysis (WGCNA), Least Absolute Shrinkage and Selection Operator (LASSO), univariate and multivariate Cox regression analyses for prognostic gene screening. Ultimately, we identified seven PCD-related genes to establish the PCD-related risk score for the advanced NSCLC model (PRAN), effectively stratifying overall survival (OS) in patients with advanced NSCLC. Multivariate Cox regression analysis revealed that the PRAN was the independent prognostic factor than clinical baseline factors. It was positively related to specific metabolic pathways, including hexosamine biosynthesis pathways, which play crucial roles in reprogramming cancer cell metabolism. Furthermore, drug prediction for different PRAN risk groups identified several sensitive drugs explicitly targeting the cell death pathway. Molecular docking analysis suggested the potential therapeutic efficacy of navitoclax in NSCLC, as it demonstrated strong binding with the amino acid residues of C-C motif chemokine ligand 14 (CCL14), carboxypeptidase A3 (CPA3), and C-X3-C motif chemokine receptor 1 (CX3CR1) proteins. The PRAN provides a robust personalized treatment and survival assessment tool in advanced NSCLC patients. Furthermore, identifying sensitive drugs for distinct PRAN risk groups holds promise for advancing targeted therapies in NSCLC.

Overexpression of Fatty Acid Synthase Upregulates Glutamine-Fructose-6-Phosphate Transaminase 1 and O-Linked N-Acetylglucosamine Transferase to Increase O-GlcNAc Protein Glycosylation and Promote Colorectal Cancer Growth.

Fatty acid synthesis has been extensively investigated as a therapeutic target in cancers, including colorectal cancer (CRC). Fatty acid synthase (FASN), a key enzyme of de novo lipid synthesis, is significantly upregulated in CRC, and therapeutic approaches of targeting this enzyme are currently being tested in multiple clinical trials. However, the mechanisms behind the pro-oncogenic action of FASN are still not completely understood. Here, for the first time, we show that overexpression of FASN increases the expression of glutamine-fructose-6-phosphate transaminase 1 (GFPT1) and O-linked N-acetylglucosamine transferase (OGT), enzymes involved in hexosamine metabolism, and the level of O-GlcNAcylation in vitro and in vivo. Consistently, expression of FASN significantly correlates with expression of GFPT1 and OGT in human CRC tissues. shRNA-mediated downregulation of GFPT1 and OGT inhibits cellular proliferation and the level of protein O-GlcNAcylation in vitro, and knockdown of GFPT1 leads to a significant decrease in tumor growth and metastasis in vivo. Pharmacological inhibition of GFPT1 and OGT leads to significant inhibition of cellular proliferation and colony formation in CRC cells. In summary, our results show that overexpression of FASN increases the expression of GFPT1 and OGT as well as the level of protein O-GlcNAcylation to promote progression of CRC; targeting the hexosamine biosynthesis pathway could be a therapeutic approach for this disease.

The GFPT2-O-GlcNAcylation-YBX1 axis promotes IL-18 secretion to regulate the tumor immune microenvironment in pancreatic cancer

The immunosuppressive microenvironment caused by several intrinsic and extrinsic mechanism has brought great challenges to the immunotherapy of pancreatic cancer. We identified GFPT2, the key enzyme in hexosamine biosynthesis pathway (HBP), as an immune-related prognostic gene in pancreatic cancer using transcriptome sequencing and further confirmed that GFPT2 promoted macrophage M2 polarization and malignant phenotype of pancreatic cancer. HBP is a glucose metabolism pathway leading to the generation of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), which is further utilized for protein O-GlcNAcylation. We confirmed GFPT2-mediated O-GlcNAcylation played an important role in regulating immune microenvironment. Through cellular proteomics, we identified IL-18 as a key downstream of GFPT2 in regulating the immune microenvironment. Through CO-IP and protein mass spectrum, we confirmed that YBX1 was O-GlcNAcylated and nuclear translocated by GFPT2-mediated O-GlcNAcylation. Then, YBX1 functioned as a transcription factor to promote IL-18 transcription. Our study elucidated the relationship between the metabolic pathway of HBP in cancer cells and the immune microenvironment, which might provide some insights into the combination therapy of HBP vulnerability and immunotherapy in pancreatic cancer.

Loss of Cardiac PFKFB2 Drives Metabolic, Functional, and Electrophysiological Remodeling in the Heart.

Phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFK-2) is a critical glycolytic regulator responsible for upregulation of glycolysis in response to insulin and adrenergic signaling. PFKFB2, the cardiac isoform of PFK-2, is degraded in the heart in the absence of insulin signaling, contributing to diabetes-induced cardiac metabolic inflexibility. However, previous studies have not examined how the loss of PFKFB2 affects global cardiac metabolism and function. To address this, we have generated a mouse model with a cardiomyocyte-specific knockout of PFKFB2 (cKO). Using 9-month-old cKO and control mice, we characterized the impacts of PFKFB2 on cardiac metabolism, function, and electrophysiology. cKO mice have a shortened life span of 9 months. Metabolically, cKO mice are characterized by increased glycolytic enzyme abundance and pyruvate dehydrogenase activity, as well as decreased mitochondrial abundance and beta oxidation, suggesting a shift toward glucose metabolism. This was supported by a decrease in the ratio of palmitoyl carnitine to pyruvate-dependent mitochondrial respiration in cKO relative to control animals. Metabolomic, proteomic, and Western blot data support the activation of ancillary glucose metabolism, including pentose phosphate and hexosamine biosynthesis pathways. Physiologically, cKO animals exhibited impaired systolic function and left ventricular dilation, represented by reduced fractional shortening and increased left ventricular internal diameter, respectively. This was accompanied by electrophysiological alterations including increased QT interval and other metrics of delayed ventricular conduction. Loss of PFKFB2 results in metabolic remodeling marked by cardiac ancillary pathway activation. This could delineate an underpinning of pathologic changes to mechanical and electrical function in the heart.

Abstract 3719: Fasting-mimicking diet plus 2-DG drives antitumor immunity by targeting EDEM3-induced PD-L1 glycosylation

Abstract The PD-1/PD-L1 axis plays an essential role in immune escape and immunotherapy. Importantly, translated N-linked glycosylation of PD-L1 stabilizes protein, enters the cell membrane binding to PD-1, renders T cells anergic and leads to T-cell exhaustion and dysfunction. Targeting PD-L1 glycosylation as an attractive target by a rational combination of cancer immunotherapies. Cancer-associated fibroblasts (CAFs), a predominant cell component of the tumor microenvironment, exert crucial functions in promoting immune escape and immunotherapy resistance by increasing the tumor PD-L1 expression. However, whether and how CAFs upregulate the glycosylation of PD-L1 has never been reported. This study shows that CAFs can upregulate PD-L1 glycosylation and enhance PD-L1 cell surface expression via glycosyltransferase EDEM3, thus cancer immune evasion. Then, the study delves into the relationship between EDEM3 expression, prognosis and immunogenicity. We demonstrate that EDEM3 plays a role in tumor immunity during tumor progression, as evidenced by EDEM3 expression is only significantly correlated with poor prognosis in the CD8+ T cell-enriched population. In subcutaneous tumor models, we observed high cell-surface PD-L1 expression accompanied by decreased CD8+ T cell infiltration and increased M2 macrophage in immunocompetent mice transplanted with EDEM3 over-expressed CRC cells that promote resistance to PD-1/PD-L1 blockade cancer immunotherapy. By performing bioinformatic analysis, we found that the hexosamine biosynthesis pathway (HBP) is highly affected in EDEM3-upregulated CRC cancer. Subsequently, the production of the UDP-GlcNAc (the end product of HBP metabolic flow), a donor substrate for N-linked glycosylation, is significantly increased in EDEM3 over-expressed CRC cells by LC/MS. Meanwhile, glucose is confirmed to be the primary effector of PD-L1 glycosylation in CRC cells, and the glucose restriction attenuates EDEM3-modified PD-L1 glycosylation by reducing UDP-GlcNAc biosynthesis. Finally, we demonstrate that modulation of glucose metabolism through a fasting-mimicking diet (FMD) in combination with 2-deoxy glucose (2-DG) treatment enhances antitumor immunity, especially in tumors with high-EDEM3 expression that exhibit resistance to immunotherapy. We propose potential therapeutic targets for CRC that inhibit PD-L1 glycosylation through FMD+2-DG and could augment the efficacy of immune checkpoint blockers. Citation Format: Xiaoxia Liu, Shaoyong Peng, Xiaolin Wang, Huichuan Yu, Yanxin Luo. Fasting-mimicking diet plus 2-DG drives antitumor immunity by targeting EDEM3-induced PD-L1 glycosylation [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 3719.

Metabolic pathways for glucose and fructose: I synthesis and metabolism of fructose by ovine conceptuses.

Fructose, the most abundant hexose sugar in fetal fluids and blood of sheep and other ungulates and cetaceans, is synthesized from glucose via the polyol pathway in trophectoderm and chorion. However, the cell-specific and temporal expression of enzymes for the synthesis and metabolism of fructose in sheep conceptuses (embryo and placental membranes) and placentomes has not been characterized. This study characterized key enzymes involved in fructose synthesis and metabolism by ovine conceptuses throughout pregnancy. Day 17 conceptuses expressed mRNAs for the polyol pathway (SORD and AKR1B1) and glucose and fructose metabolism (HK1, HK2, G6PD, OGT, and FBP), but not those required for gluconeogenesis (G6Pase or PCK). Ovine placentomes also expressed mRNAs for SORD, AKR1B1, HK1, and OGT. Fructose can be metabolized via the ketohexokinase (KHK) pathway and isoforms, KHK-A and KHK-C, were expressed in ovine conceptuses from Day 16 of pregnancy and placentomes during pregnancy in a cell specific manner: KHK-A protein was more abundant in trophectoderm and cotyledons of placentomes, while KHK-C protein was more abundant in endoderm of Day 16 conceptuses and chorionic epithelium in placentomes. Expression of KHK mRNAs in placentomes was greatest at Day 30 of pregnancy (P < 0.05), but not different among days later in gestation. These results provide novel insights into the synthesis and metabolism of fructose via the uninhibited KHK pathway in ovine conceptuses to generate ATP via the TCA cycle, as well as substrates for the pentose cycle, hexosamine biosynthesis pathway and one-carbon metabolism required for conceptus development throughout pregnancy.

Hexosamine biosynthesis and related pathways, protein N-glycosylation and O-GlcNAcylation: their interconnection and role in plants.

N-Acetylglucosamine (GlcNAc), a fundamental amino sugar moiety, is essential for protein glycosylation, glycolipid, GPI-anchor protein, and cell wall components. Uridine diphosphate-GlcNAc (UDP-GlcNAc), an active form of GlcNAc, is synthesized through the hexosamine biosynthesis pathway (HBP). Although HBP is highly conserved across organisms, the enzymes involved perform subtly distinct functions among microbes, mammals, and plants. A complete block of HBP normally causes lethality in any life form, reflecting the pivotal role of HBP in the normal growth and development of organisms. Although HBP is mainly composed of four biochemical reactions, HBP is exquisitely regulated to maintain the homeostasis of UDP-GlcNAc content. As HBP utilizes substrates including fructose-6-P, glutamine, acetyl-CoA, and UTP, endogenous nutrient/energy metabolites may be integrated to better suit internal growth and development, and external environmental stimuli. Although the genes encoding HBP enzymes are well characterized in microbes and mammals, they were less understood in higher plants in the past. As the HBP-related genes/enzymes have largely been characterized in higher plants in recent years, in this review we update the latest advances in the functions of the HBP-related genes in higher plants. In addition, HBP's salvage pathway and GlcNAc-mediated two major co- or post-translational modifications, N-glycosylation and O-GlcNAcylation, are also included in this review. Further knowledge on the function of HBP and its product conjugates, and the mechanisms underlying their response to deleterious environments might provide an alternative strategy for agricultural biofortification in the future.

O-GlcNAc of STING mediates antiviral innate immunity

BackgroundO-GlcNAcylation modification affects multiple physiological and pathophysiolocal functions of cells. Altered O-GlcNAcylation was reported to participate in antivirus response. Stimulator of interferon genes (STING) is an adaptor mediating DNA virus-induced innate immune response. Whether STING is able to be modified by O-GlcNAcylation and how O-GlcNAcylation affects STING-mediated anti-DNA virus response remain unknown.MethodsMetabolomics analysis was used for detecting metabolic alterations in HSV-1 infection cells. Succinylated wheat germ agglutinin (sWGA), co-immunoprecipitation, and pull-down assay were employed for determining O-GlcNAcylation. Mutagenesis PCR was applied for the generation of STING mutants. WT and Sting1−/− C57BL/6 mice (KOCMP-72512-Sting1-B6NVA) were infected with HSV-1 and treated with O-GlcNAcylation inhibitor for validating the role of STING O-GlcNAcylation in antiviral response.ResultsSTING was functionally activated by O-GlcNAcylation in host cells challenged with HSV-1. We demonstrated that this signaling event was initiated by virus infection-enhanced hexosamine biosynthesis pathway (HBP). HSV-1 (or viral DNA mimics) promotes glucose metabolism of host cells with a marked increase in HBP, which provides donor glucosamine for O-GlcNAcylation. STING was O-GlcNAcylated on threonine 229, which led to lysine 63-linked ubiquitination of STING and activation of antiviral immune responses. Mutation of STING T229 to alanine abrogated STING activation and reduced HSV-1 stimulated production of interferon (IFN). Application of 6-diazo-5-oxonorleucine (DON), an agent that blocks the production of UDP-GlcNAc and inhibits O-GlcNAcylation, markedly attenuated the removal of HSV-1 in wild type C57BL/6 mice, leading to an increased viral retention, elevated infiltration of inflammatory cells, and worsened tissue damages to those displayed in STING gene knockout mice. Together, our data suggest that STING is O-GlcNAcylated in HSV-1, which is crucial for an effective antiviral innate immune response.ConclusionHSV-1 infection activates the generation of UDP-Glc-NAc by upregulating the HBP metabolism. Elevated UDP-Glc-NAc promotes the O-GlcNAcylation of STING, which mediates the anti-viral function of STING. Targeting O-GlcNAcylation of STING could be a useful strategy for antiviral innate immunity.

Discovery of metal-binding proteins by thermal proteome profiling.

Metal-binding proteins (MBPs) have various and important biological roles in all living species and many human diseases are intricately linked to dysfunctional MBPs. Here, we report a chemoproteomic method named 'metal extraction-triggered agitation logged by thermal proteome profiling' (METAL-TPP) to globally profile MBPs in proteomes. The method involves the extraction of metals from MBPs using chelators and monitoring the resulting protein stability changes through thermal proteome profiling. Applying METAL-TPP to the human proteome with a broad-spectrum chelator, EDTA, revealed a group of proteins with reduced thermal stability that contained both previously known MBPs and currently unannotated MBP candidates. Biochemical characterization of one potential target, glutamine-fructose-6-phosphate transaminase 2 (GFPT2), showed that zinc bound the protein, inhibited its enzymatic activity and modulated the hexosamine biosynthesis pathway. METAL-TPP profiling with another chelator, TPEN, uncovered additional MBPs in proteomes. Collectively, this study developed a robust tool for proteomic discovery of MBPs and provides a rich resource for functional studies of metals in cell biology.

FpOGT is required for fungal growth, stress response, and virulence of Fusarium proliferatum by affecting the expression of glucokinase and other glucose metabolism-related genes

O-GlcNAcylation, an important post-translational modification catalyzed by O-GlcNAc transferase (OGT), plays critical roles in several biological processes. In this study, we present our findings on the function of FpOGT in regulating physiological processes and pathogenicity of Fusarium proliferatum (Fp), the alfalfa root rot fungus. The deletion of FpOGT impaired mycelial growth and altered macroconidia morphology in Fp. Furthermore, ΔFpOGT mutant displayed altered tolerance to various stressors, including cell wall perturbing agents, osmotic stressors, metal ionic stressors, and fungicides. Deletion of FpOGT significantly decreased Fp virulence toward alfalfa. The transcriptome analysis demonstrated that FpOGT plays a regulatory role in glucose metabolic pathways, including glycolysis, tricarboxylic acid (TCA) cycle, and hexosamine biosynthesis pathway (HBP), by influencing the expression of relevant genes. The downregulation of the glucokinase gene, FpGCK, was observed in ΔFpOGT, and the disruption of FpGCK led to a decrease in Fp virulence. Additionally, FpOGT affected the expression levels of the FpGCK-AS1 isoform, thereby impacting glucokinase function. The molecular docking analysis elucidated the plausible physical interaction between FpOGT and FpGCK, thereby offering valuable insights into their interrelationship. These findings underscore the indispensable involvement of FpOGT, the sole O-GlcNAc transferase in Fp, in various biological processes and the pathogenicity through its regulation of fundamental metabolic processes. Consequently, this study emphasizes the significance and elucidates the molecular mechanism underlying the role of O-GlcNAc transferase in diverse fundamental biological processes and the pathogenicity of phytopathogenic fungi.

Protein O-GlcNAcylation: The sweet hub in liver metabolic flexibility from a (patho)physiological perspective.

O-GlcNAcylation is a dynamic, reversible and atypical O-glycosylation that regulates various cellular physiological processes via conformation, stabilisation, localisation, chaperone interaction or activity of target proteins. The O-GlcNAcylation cycle is precisely controlled by collaboration between O-GlcNAc transferase and O-GlcNAcase. Uridine-diphosphate-N-acetylglucosamine, the sole donor of O-GlcNAcylation produced by the hexosamine biosynthesis pathway, is controlled by the input of glucose, glutamine, acetyl coenzyme A and uridine triphosphate, making it a sensor of the fluctuation of molecules, making O-GlcNAcylation a pivotal nutrient sensor for the metabolism of carbohydrates, amino acids, lipids and nucleotides. O-GlcNAcylation, particularly prevalent in liver, is the core hub for controlling systemic glucose homeostasis due to its nutritional sensitivity and precise spatiotemporal regulation of insulin signal transduction. The pathology of various liver diseases has highlighted hepatic metabolic disorder and dysfunction, and abnormal O-GlcNAcylation also plays a specific pathological role in these processes. Therefore, this review describes the unique features of O-GlcNAcylation and its dynamic homeostasis maintenance. Additionally, it explains the underlying nutritional sensitivity of O-GlcNAcylation and discusses its mechanism of spatiotemporal modulation of insulin signal transduction and liver metabolic homeostasis during the fasting and feeding cycle. This review emphasises the pathophysiological implications of O-GlcNAcylation in nonalcoholic fatty liver disease, nonalcoholic steatohepatitis and hepatic fibrosis, and focuses on the adverse effects of hyper O-GlcNAcylation on liver cancer progression and metabolic reprogramming.

Pancreatic Cancer: An Exocrine Tumor with Endocrine Characteristics.

To examine the characteristics of pancreatic cancer patients with long-term survival. Although pancreatic cancer is a highly lethal malignancy, a minority of patients experience long-term survival. The characteristics of these patients remain largely unidentified. An indolent subgroup was established using carbohydrate antigen 19-9 (CA19-9), which is the best-validated biomarker for pancreatic cancer. Of 1558 patients, 13.9% were included in the CA19-9-normal (≤ 37U/mL) subgroup. A normal A19-9 level was an independent variable for overall survival (median survival, 18.1 vs. 9.7 months, hazard ratio = 0.53, P < 0.001). The 5-year survival of patients with stage IV CA19-9-normal cancer was higher than that of patients with stage I-IV CA19-9-high cancer (22.4% vs. 6.8%, P = 0.034). The CA19-9-normal subgroup exhibited reduced levels of circulating glucose (P < 0.001) and increased expression of insulin (P < 0.001) compared with the CA19-9-high subgroup. Glucose was a substrate for CA19-9 biosynthesis through the hexosamine biosynthesis pathway. In addition, in pancreatic cancer animal models of diabetes, glucose control decreased CA19-9 levels and improved overall survival. In a clinical trial (NCT05306028) of patients before undergoing major anticancer treatments, glucose control decreased CA19-9 levels in 90.9% of the patients. CA19-9-normal pancreatic cancer is a strikingly indolent subgroup with low glucose and high insulin. Glucose control is a promising therapeutic strategy for pancreatic cancer.

GFPT1 deficiency is a novel cause of a dilated cardiomyopathy and is required for myocardial adaptation to chronic pressure-overload stress-induced heart failure

Abstract Background GFPT1 is the rate-limiting enzyme of a pathway called the hexosamine biosynthesis pathway (HBP) that turns fructose 6-phosphate derived from glucose into another monosaccharide called N-acetylglucosamine (GlcNAc). GlcNAc is an important substrate for modifying diverse cellular proteins on their serine and threonine residues termed O-GlcNAcylation often competing with phosphorylation and altering their activity or stability (Bond &amp; Hanover; 2015). Increased GFPT1 activity is known to stimulate the cellular integrated stress response improving protein quality control and cell survival (Denzel et al 2014). In the heart, GFPT1 expression and O-GlcNAcylation increases in the early stages of chronic cardiac stress but the physiological role of GFPT1 in regulating heart function including its role during chronic stress remains unclear. Identifying molecular pathways that promote adaptive cardiac remodelling is a promising approach to discover novel therapies for heart failure. Methods Cardiomyocyte-specific GFPT1 knockout mice were generated to study the effects of GFPT1 deletion in hearts. Heart function was examined by echocardiography. HBP activity was quantified using Western blotting of O-GlcNAc-modified proteins. Abdominal aortic banding (AAB) was used to induce chronic pressure-overload heart failure. Results Homozygous GFPT1 KO mice spontaneously develop a dilated cardiomyopathy (DCM) with a 30% mortality rate (ejection fraction 32% compared with 62% for floxed controls and end diastolic volume 125 microL versus 60 microL; n=5-6 per group; p &amp;lt;0.05). Hearts showed reduced levels of total protein O-GlcNAcylation suggesting impaired hexosamine biosynthesis pathway activity. Heart function could be rescued by supplementing their diets with oral glucosamine bypassing GFPT1 to provide metabolites required for hexosamine biosynthesis and normalising O-GlcNAcylation (ejection fraction improved to 40% in KO with supplementation versus KO without; n=4-6 per group; p &amp;lt;0.05). Heterozygous KO mice do not develop a DCM but when subject to pressure-overload cardiac stress, develop worse heart failure with a 50% mortality rate over 9 weeks of pressure-overload (ejection fraction 43% in het KO versus 53% in floxed control; n= 3-6 per group; p = 0.0025). Conclusion Deficiency of GFPT1 is a novel cause of a spontaneous dilated cardiomyopathy. GFPT1 is required for physiological growth of the heart that may in part involve the O-GlcNAcylation of proteins. Partial GFPT1 deficiency is associated with adverse cardiac remodelling following pressure-overload stress. Increasing GFPT1 activity may be a key therapeutic target in developing novel heart failure treatments.

O-GlcNAc transferase promotes glioblastoma by modulating genes responsible for cell survival, invasion, and inflammation

Metabolic reprogramming has emerged as one of the key hallmarks of cancer cells. Various metabolic pathways are dysregulated in cancers, including the hexosamine biosynthesis pathway (HBP). Protein O-GlcNAcylation is catalyzed by the enzyme O-GlcNAc transferase (OGT), an effector of HBP that is found to be upregulated in most cancers. Post-translational O-GlcNAcylation of various signaling and transcriptional regulators could promote cancer cell maintenance and progression by regulating gene expression, as gene-specific transcription factors and chromatin regulators are among the most highly O-GlcNAcylated proteins. Here, we investigated the role of OGT in glioblastoma. We demonstrate OGT knockdown and chemical inhibition led to reduced glioblastoma cell proliferation and downregulation of many genes known to play key roles in glioblastoma cell proliferation, migration, and invasion. We show genes downregulated due to OGT reduction are also known to be transcriptionally regulated by transcriptional initiation/elongation co-factor BRD4. We found BRD4 to be O-GlcNAcylated in glioblastoma cells, however OGT knockdown/inhibition neither changed its expression nor its chromatin association on promoters. Intriguingly, we observed OGT knockdown led to reduced Pol II-Ser2P chromatin association on target genes independent of BRD4 and without affecting other transcription initiation/elongation factors. Finally, we found chemical inhibition of BRD4 potentiated the effects of OGT inhibition in reducing glioblastoma cell proliferation, invasion, and migration. We propose BRD4 and OGT act independently in the transcriptional regulation of a common set of genes and that combined inhibition of OGT and BRD4 could be utilized therapeutically for more efficient glioblastoma cell targeting than targeting of either protein alone.

MAVS integrates glucose metabolism and RIG-I-like receptor signaling

MAVS is an adapter protein involved in RIG-I-like receptor (RLR) signaling in mitochondria, peroxisomes, and mitochondria-associated ER membranes (MAMs). However, the role of MAVS in glucose metabolism and RLR signaling cross-regulation and how these signaling pathways are coordinated among these organelles have not been defined. This study reports that RLR action drives a switch from glycolysis to the pentose phosphate pathway (PPP) and the hexosamine biosynthesis pathway (HBP) through MAVS. We show that peroxisomal MAVS is responsible for glucose flux shift into PPP and type III interferon (IFN) expression, whereas MAMs-located MAVS is responsible for glucose flux shift into HBP and type I IFN expression. Mechanistically, peroxisomal MAVS interacts with G6PD and the MAVS signalosome forms at peroxisomes by recruiting TNF receptor-associated factor 6 (TRAF6) and interferon regulatory factor 1 (IRF1). By contrast, MAMs-located MAVS interact with glutamine-fructose-6-phosphate transaminase, and the MAVS signalosome forms at MAMs by recruiting TRAF6 and TRAF2. Our findings suggest that MAVS mediates the interaction of RLR signaling and glucose metabolism.

Small extracellular vesicles delivering lncRNA WAC-AS1 aggravate renal allograft ischemia‒reperfusion injury by inducing ferroptosis propagation.

Ferroptosis is a predominant contributor to renal ischemia reperfusion injury (IRI) after kidney transplant, evoking delayed graft function and poorer long-term outcomes. The wide propagation of ferroptosis among cell populations in a wave-like manner, developing the "wave of ferroptosis" causes a larger area of tubular necrosis and accordingly aggravates renal allograft IRI. In this study, we decipher a whole new metabolic mechanism underlying ferroptosis and propose a novel spreading pathway of the "wave of ferroptosis" in the renal tissue microenvironment, in which renal IRI cell-secreted small extracellular vesicles (IRI-sEVs) delivering lncRNA WAC-AS1 reprogram glucose metabolism in adjacent renal tubular epithelial cell populations by inducing GFPT1 expression and increasing hexosamine biosynthesis pathway (HBP) flux, and consequently enhances O-GlcNAcylation. Additionally, BACH2 O-GlcNAcylation at threonine 389 in renal tubular epithelial cells prominently inhibits its degradation by ubiquitination and promotes importin α5-mediated nuclear translocation. We present the first evidence that intranuclear BACH2 suppresses SLC7A11 and GPX4 transcription by binding to their proximal promoters and decreases cellular anti-peroxidation capability, accordingly facilitating ferroptosis. Inhibition of sEV biogenesis and secretion by GW4869 and knockout of lncRNA WAC-AS1 in IRI-sEVs both unequivocally diminished the "wave of ferroptosis" propagation and protected against renal allograft IRI. The functional and mechanistic regulation of IRI-sEVs was further corroborated in an allograft kidney transplant model and an in situ renal IRI model. In summary, these findings suggest that inhibiting sEV-mediated lncRNA WAC-AS1 secretion and targeting HBP metabolism-induced BACH2 O-GlcNAcylation in renal tubular epithelial cells may serve as new strategies for protecting against graft IRI after kidney transplant.

Foetal recapitulation of nutrient surplus signalling by O-GlcNAcylation and the failing heart.

The development of the foetal heart is driven by increased glucose uptake and activation of mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1α (HIF-1α), which drives glycolysis. In contrast, the healthy adult heart is governed by sirtuin-1 (SIRT1) and adenosine monophosphate-activated protein kinase (AMPK), which promote fatty-acid oxidation and the substantial mitochondrial ATP production required for survival in a high-workload normoxic environment. During cardiac injury, the heart recapitulates the foetal signalling programme, which (although adaptive in the short term) is highly deleterious if sustained for long periods of time. Prolonged increases in glucose uptake in cardiomyocytes under stress leads to increased flux through the hexosamine biosynthesis pathway; its endproduct-uridine diphosphate N-acetylglucosamine (UDP-GlcNAc)-functions as a critical nutrient surplus sensor. UDP-GlcNAc drives the post-translational protein modification known as O-GlcNAcylation, which rapidly and reversibly modifies thousands of intracellular proteins. Both O-GlcNAcylation and phosphorylation act at serine/threonine residues, but whereas phosphorylation is regulated by hundreds of specific kinases and phosphatases, O-GlcNAcylation is regulated by only two enzymes, O-GlcNAc transferase (OGT) and O-GlcNAcase (OGA), which adds or removes GlcNAc (N-acetylglucosamine), respectively, from target proteins. Recapitulation of foetal programming in heart failure (regardless of diabetes) is accompanied by marked increases in O-GlcNAcylation, both experimentally and clinically. Heightened O-GlcNAcylation in the heart leads to impaired calcium kinetics and contractile derangements, arrhythmias related to activation of voltage-gated sodium channels and Ca2+ /calmodulin-dependent protein kinase II, mitochondrial dysfunction, and maladaptive hypertrophy, microvascular dysfunction, fibrosis and cardiomyopathy. These deleterious effects can be prevented by suppression of O-GlcNAcylation, which can be achieved experimentally by upregulation of AMPK and SIRT1 or by pharmacological inhibition of OGT or stimulation of OGA. The effects of sodium-glucose cotransporter 2 (SGLT2) inhibitors on the heart are accompanied by reduced O-GlcNAcylation, and their cytoprotective effects are reportedly abrogated if their action to suppress O-GlcNAcylation is blocked. Such an action may represent one of the many mechanisms by which enhanced AMPK and SIRT1 signalling following SGLT2 inhibition leads to cardiovascular benefits. These observations, taken collectively, suggest that UDP-GlcNAc functions as a critical nutrient surplus sensor (which acting in concert with mTOR and HIF-1α) can promote the development of cardiomyopathy.

Roles of GFAT and PFK genes in energy metabolism of brown planthopper, Nilaparvata lugens.

Glutamine:fructose-6-phosphate aminotransferases (GFATs) and phosphofructokinase (PFKs) are the principal rate-limiting enzymes involved in hexosamine biosynthesis pathway (HBP) and glycolysis pathway, respectively. In this study, the NlGFAT and NlPFK were knocked down through RNA interference (RNAi) in Nilaparvata lugens, the notorious brown planthopper (BPH), and the changes in energy metabolism were determined. Knockdown of either NlGFAT or NlPFK substantially reduced gene expression related to trehalose, glucose, and glycogen metabolism pathways. Moreover, trehalose content rose significantly at 72h after dsGFAT injection, and glycogen content increased significantly at 48h after injection. Glucose content remained unchanged throughout the experiment. Conversely, dsPFK injection did not significantly alter trehalose, but caused an extreme increase in glucose and glycogen content at 72h after injection. The Knockdown of NlGFAT or NlPFK significantly downregulated the genes in the glycolytic pathway, as well as caused a considerable and significant decrease in pyruvate kinase (PK) activity after 48h and 72h of inhibition. After dsGFAT injection, most of genes in TCA cycle pathway were upregulated, but after dsNlPFK injection, they were downregulated. Correspondingly, ATP content substantially increased at 48h after NlGFAT knockdown but decreased to an extreme extent by 72h. In contrast, ATP content decreased significantly after NlPFK was knocked down and returned. The results have suggested the knockdown of either NlGFAT or NlPFK resulted in metabolism disorders in BPHs, highlighting the difference in the impact of those two enzyme genes on energy metabolism. Given their influence on BPHs energy metabolism, developing enzyme inhibitors or activators may provide a biological control for BPHs.

Fetal Reprogramming of Nutrient Surplus Signaling, O-GlcNAcylation, and the Evolution of Chronic Kidney Disease.

Fetal kidneys have increased uptake of glucose, ATP production by glycolysis, and upregulation of mammalian target of rapamycin (mTOR) and hypoxia-inducible factor-1α (HIF-1α). These interacting processes promote nephrogenesis in a hypoxic low-tubular-workload environment. In contrast, the healthy adult kidney upregulates sirtuin-1 and adenosine monophosphate-activated protein kinase, which enhances ATP production through fatty-acid oxidation, in a normoxic high-tubular-workload environment. During stress or injury, the kidney reverts to a fetal signaling program that is adaptive in the short-term, but deleterious if sustained with heightened oxygen tension and tubular workloads. Prolonged increase in glucose uptake in glomerular and proximal tubular cells leads to enhanced flux through the hexosamine biosynthesis pathway. The endproduct - uridine diphosphate N -acetylglucosamine (UDP-GlcNAc) - drives the rapid and reversible O-GlcNAcylation of thousands of intracellular proteins, most not membrane-bound or secreted. O-GlcNAcylation and phosphorylation act at serine/threonine residues, but whereas hundreds of kinases and phosphatases regulate phosphorylation, only O-GlcNAc transferase and O-GlcNAcase, which add or remove O-GlcNAc from target proteins, regulate O-GlcNAcylation. Fetal reprogramming (upregulating mTOR and HIF-1α) and increased O-GlcNAcylation occur in diabetic and nondiabetic CKD, experimentally and clinically. Augmentation of O-GlcNAcylation in the adult kidney enhances oxidative stress, cell cycle entry, and apoptosis, activates proinflammatory and profibrotic pathways, and inhibits megalin-mediated albumin endocytosis in glomerular mesangial and proximal tubular cells, which muting of O-GlcNAcylation can aggravate and attenuate, respectively. Additionally, some nephroprotective drugs are associated with diminished O-GlcNAcylation in the kidney. Evidence supports further investigation of UDP-GlcNAc as a critical nutrient surplus sensor in the development of diabetic and nondiabetic CKD.

Pharmacological inhibition of lipolysis prevents adverse metabolic outcomes during glucocorticoid administration

ObjectiveGlucocorticoids are one of the most commonly prescribed classes of anti-inflammatory drugs; however, chronic treatment promotes iatrogenic (drug-induced) diabetes. As part of their physiological role, glucocorticoids stimulate lipolysis to spare glucose. We hypothesized that persistent stimulation of lipolysis during glucocorticoid therapy plays a causative role in the development of iatrogenic diabetes. MethodsMale C57BL/6J mice were given 100 μg/mL corticosterone (Cort) in the drinking water for two weeks and were fed either normal chow (TekLad 8640) or the same diet supplemented with an adipose triglyceride lipase inhibitor (Atglistatin - 2 g/kg diet) to inhibit the first step of lipolysis. ResultsHerein, we report for the first time that glucocorticoid administration promotes a unique state of substrate excess and energetic overload in skeletal muscle that primarily results from the rampant mobilization of endogenous fuels. Inhibiting lipolysis protected mice from Cort-induced gains in fat mass, excess ectopic lipid accrual, hyperinsulinemia, and hyperglycemia. The role lipolysis plays in Cort-mediated pathology appears to differ between tissues. Within skeletal muscle, Cort-induced lipolysis facilitated diversion of glucose-derived carbons toward the pentose phosphate and hexosamine biosynthesis pathways but contributed to <3% of the Cort-induced genomic adaptations. In contrast, Cort stimulation of lipolysis accounted for ∼35% of the genomic changes in the liver but had minimal impact on hepatic metabolites reported. ConclusionsThese data support the idea that activation of lipolysis plays a causal role in the progression toward iatrogenic diabetes during glucocorticoid therapy with differential impact on skeletal muscle and liver.