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Rewired metabolism is a hallmark of pancreatic ductal adenocarcinomas (PDA). Previously, we demonstrated that PDA cells enhance glycosylation precursor biogenesis through the hexosamine biosynthetic pathway (HBP) via activation of the rate limiting enzyme, glutamine-fructose 6-phosphate amidotransferase 1 (GFAT1). Here, we genetically ablated GFAT1 in human PDA cell lines, which completely blocked proliferation in vitro and led to cell death. In contrast, GFAT1 knockout did not preclude the growth of human tumor xenografts in mice, suggesting that cancer cells can maintain fidelity of glycosylation precursor pools by scavenging nutrients from the tumor microenvironment. We found that hyaluronic acid (HA), an abundant carbohydrate polymer in pancreatic tumors composed of repeating N-acetyl-glucosamine (GlcNAc) and glucuronic acid sugars, can bypass GFAT1 to refuel the HBP via the GlcNAc salvage pathway. Together, these data show HA can serve as a nutrient fueling PDA metabolism beyond its previously appreciated structural and signaling roles.

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.

Pancreatic ductal adenocarcinoma (PDAC) is a leading cause of cancer-related death and the search for a resolutive therapy is still a challenge. Since KRAS is commonly mutated in PDAC and is one of the main drivers of PDAC progression, its inhibition should be a key strategy for treatment, especially considering the recent development of specific KRAS inhibitors. Nevertheless, the effects of KRAS inhibition can be increased through the co-inhibition of other nodes important for cancer development. One of them could be the hexosamine biosynthetic pathway (HBP), whose enhancement is considered fundamental for PDAC. Here, we demonstrate that PDAC cells expressing oncogenic KRAS, owing to an increase in the HBP flux, become strongly reliant on HBP for both proliferation and survival. In particular, upon treatment with two different compounds, 2-deoxyglucose and FR054, inhibiting both HBP and protein N-glycosylation, these cells undergo apoptosis significantly more than PDAC cells expressing wild-type KRAS. Importantly, we also show that the combined treatment between FR054 and the pan-RAS inhibitor BI-2852 has an additive negative effect on cell proliferation and survival by means of the suppression of both Akt activity and cyclin D1 expression. Thus, co-inhibition of HBP and oncogenic RAS may represent a novel therapy for PDAC patients.

Rationale: Viruses hijack the host cell machinery to promote viral replication; however, the mechanism by which metabolic reprogramming regulates innate antiviral immunity in the host remains elusive. Herein, we explore how the hexosamine biosynthesis pathway (HBP) and O-linked-N-acetylglucosaminylation (O-GlcNAcylation) regulate host antiviral response against hepatitis B virus (HBV) in vitro and in vivo. Methods: We conducted a metabolomics assay to evaluate metabolic responses of host cells to HBV infection. We systematically explored the role of HBP and protein O-GlcNAcylation in regulating HBV infection in cell and mouse models. O-linked N-acetylglucosamine (O-GlcNAc) target proteins were identified via liquid chromatography-tandem mass spectrometry (LC-MS) and co-immunoprecipitation assays. Additionally, we also examined uridine diphosphate (UDP)-GlcNAc biosynthesis and O-GlcNAcylation levels in patients with chronic hepatitis B (CHB). Results: HBV infection upregulated GLUT1 expression on the hepatocyte surface and facilitated glucose uptake, which provides substrates to HBP to synthesize UDP-GlcNAc, leading to an increase in protein O-GlcNAcylation. Pharmacological or transcriptional inhibition of HBP and O-GlcNAcylation promoted HBV replication. Mechanistically, O-GlcNAc transferase (OGT)-mediated O-GlcNAcylation of sterile alpha motif and histidine/aspartic acid domain-containing protein 1 (SAMHD1) on Ser93 stabilizes SAMHD1 and enhances its antiviral activity. Analysis of clinical samples revealed that UDP-GlcNAc level was increased, and SAMHD1 was O-GlcNAcylated in patients with CHB. Conclusions: HBP-mediated O-GlcNAcylation positively regulates host antiviral response against HBV in vitro and in vivo. The findings reveal a link between HBP, O-GlcNAc modification, and innate antiviral immunity by targeting SAMHD1.

The proteasome inhibitor bortezomib (BTZ) has emerged as an effective drug for the treatment of multiple myeloma even though many patients relapse from BTZ therapy. The present study investigated the metabolic pathways underlying the acquisition of bortezomib resistance in multiple myeloma. We used two different clones of multiple myeloma cell lines exhibiting different sensitivities to BTZ (U266 and U266-R) and compared them in terms of metabolic profile, mitochondrial fitness and redox balance homeostasis capacity. Our results showed that the BTZ-resistant clone (U266-R) presented increased glycosylated UDP-derivatives when compared to BTZ-sensitive cells (U266), thus also suggesting higher activities of the hexosamine biosynthetic pathway (HBP), regulating not only protein O- and N-glycosylation but also mitochondrial functions. Notably, U266-R displayed increased mitochondrial biogenesis and mitochondrial dynamics associated with stronger antioxidant defenses. Furthermore, U266-R maintained a significantly higher concentration of substrates for protein glycosylation when compared to U266, particularly for UDP-GlcNac, thus further suggesting the importance of glycosylation in the BTZ pharmacological response. Moreover, BTZ-treated U266-R showed significantly higher ATP/ADP ratios and levels of ECP and also exhibited increased mitochondrial fitness and antioxidant response. In conclusions, our findings suggest that the HBP may play a major role in mitochondrial fitness, driving BTZ resistance in multiple myeloma and thus representing a possible target for new drug development for BTZ-resistant patients.

Summary The mouse embryo undergoes compaction at the 8-cell stage, and its transition to 16 cells generates polarity such that the outer apical cells are trophectoderm (TE) precursors and the inner cell mass (ICM) gives rise to the embryo. Here, we report that this first cell fate specification event is controlled by glucose. Glucose does not fuel mitochondrial ATP generation, and glycolysis is dispensable for blastocyst formation. Furthermore, glucose does not help synthesize amino acids, fatty acids, and nucleobases. Instead, glucose metabolized by the hexosamine biosynthetic pathway (HBP) allows nuclear localization of YAP1. In addition, glucose-dependent nucleotide synthesis by the pentose phosphate pathway (PPP), along with sphingolipid (S1P) signaling, activates mTOR and allows translation of Tfap2c. YAP1, TEAD4, and TFAP2C interact to form a complex that controls TE-specific gene transcription. Glucose signaling has no role in ICM specification, and this process of developmental metabolism specifically controls TE cell fate.

McArdle disease, also known as glycogen storage disease type V (GSDV), is characterized by exercise intolerance, the second wind phenomenon, and high serum creatine kinase activity. Here, we recapitulate PYGM mutations in the population responsible for this disease. Traditionally, McArdle disease has been considered a metabolic myopathy caused by the lack of expression of the muscle isoform of the glycogen phosphorylase (PYGM). However, recent findings challenge this view, since it has been shown that PYGM is present in other tissues than the skeletal muscle. We review the latest studies about the molecular mechanism involved in glycogen phosphorylase activity regulation. Further, we summarize the expression and functional significance of PYGM in other tissues than skeletal muscle both in health and McArdle disease. Furthermore, we examine the different animal models that have served as the knowledge base for better understanding of McArdle disease. Finally, we give an overview of the latest state-of-the-art clinical trials currently being carried out and present an updated view of the current therapies. Fil: Llavero, Francisco. Universidad del País Vasco; España. Universidad Europea de Madrid; España Fil: Sastre, Alazne Arrazola. Universidad del País Vasco; España Fil: Montoro, Miriam Luque. Universidad del País Vasco; España Fil: Gálvez, Patricia. Universidad del País Vasco; España. Parque Tecnológico de Ciencias de la Salud; España Fil: Lacerda, Hadriano M.. Universidad del País Vasco; España Fil: Parada, Luis Antonio. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Salta. Instituto de Patología Experimental. Universidad Nacional de Salta. Facultad de Ciencias de la Salud. Instituto de Patología Experimental; Argentina Fil: Zugaza, José Luis. Universidad del País Vasco; España. Fundación Vasca para la Ciencia; España

The Hexosamine Biosynthetic Pathway (HBP) is a branch of glycolysis responsible for the production of a key substrate for protein glycosylation, UDP-GlcNAc. Cancer cells present altered glucose metabolism and aberrant glycosylation, pointing to alterations on HBP. Recently it was demonstrated that HBP influences many aspects of tumor biology, including the development of metastasis. In this work we characterize HBP in melanoma cells and analyze its importance to cellular processes related to the metastatic phenotype. We demonstrate that an increase in HBP flux, as well as increased O-GlcNAcylation, leads to decreased cell motility and migration in melanoma cells. In addition, inhibition of N- and O-glycosylation glycosylation reduces cell migration. High HBP flux and inhibition of N-glycosylation decrease the activity of metalloproteases 2 and 9. Our data demonstrates that modulation of HBP and different types of glycosylation impact cell migration.

Purpose The severity of untreated or refractory diabetes mellitus has been functionally linked to elevated concentrations of free plasma glucose, clinically defined as hyperglycemia. Operationally, the pathophysiological presentations of prolonged hyperglycemia may be categorized within insulin-dependent and insulin-independent, type 1 and type 2 diabetic phenotypes, respectively. Accordingly, major areas of empirical biomedical research have focused on the elucidation of underlying mechanisms driving key cellular signaling systems that are significantly altered in patients presenting with diabetes-associated chronic hyperglycemia. Methods Presently, we provide a translationally oriented review of key studies evaluating the aberrant effects of hyperglycemia on two major signaling pathways linked to debilitating cellular and systemic effects via targeted disruption of mitochondrial bioenergetics: (1) advanced glycation end-products (AGEs)/and their cognate receptor for advanced glycation end-products (RAGEs), and (2) the hexosamine biosynthetic pathway (HBP). Results In preclinical models, cultured vascular endothelial cells exposed to hyperglycemic glucose concentrations were observed to produce enhanced levels of reactive oxygen species (ROS) functionally linked to increased formation of AGEs and expression of their cognate RAGEs. Importantly, inhibitors of AGEs formation, mitochondrial complex II, or un-couplers of oxidative phosphorylation, were observed to significantly reduce the effects of hyperglycemia on ROS production and cellular damage, thereby establishing a critical linkage to multiple levels of mitochondrial functioning. Hyperglycemia-mediated enhancement of mitochondrial ROS/superoxide production in vascular endothelial cells has been functionally linked to the shunting of glucose into the HBP with resultant long-term activation of pro-inflammatory signaling processes. Additionally, exposure of cultured cells to hyperglycemic conditions resulted in enhanced HBP-mediated inhibition of protein subunits of mitochondrial respiratory complexes I, III, and IV, intimately associated with normative cellular bioenergetics and ATP production. Conclusions Convergent lines of evidence link chronic hyperglycemic conditions to aberrant expression of AGEs/RAGEs and HBP signaling pathways in relation to the pathophysiological formation of ROS and pro-inflammatory processes on the functional dysregulation of mitochondrial bioenergetics.

We have uncovered that epithelial plasticity programs metabolically reprogram epithelial lung cells by increasing expression of genes (e.g., glutamine-fructose-6-phosphate transaminase 2 – GFPT2 and UDP-N-acetylglucosamine pyrophosphorylase 1 – UAP1) critical for the hexosamine biosynthetic pathway (HBP) and elevating global protein O-GlcNAcylation – a specific type of glycosylation. We found that increased O-GlcNAcylation could suppress oncogene-induced senescence tumor suppressor pathways that ultimately led to accelerated KrasG12D-driven lung tumorigenesis.

Cancer aberrant N- and O-linked protein glycosylation, frequently resulting from an augmented flux through the Hexosamine Biosynthetic Pathway (HBP), play different roles in tumor progression. However, the low specificity and toxicity of the existing HBP inhibitors prevented their use for cancer treatment. Here we report the preclinical evaluation of FR054, a novel inhibitor of the HBP enzyme PGM3, with a remarkable anti-breast cancer effect. In fact, FR054 induces in different breast cancer cells a dramatic decrease in cell proliferation and survival. In particular, in a model of Triple Negative Breast Cancer (TNBC) cells, MDA-MB-231, we show that these effects are correlated to FR054-dependent reduction of both N- and O-glycosylation level that cause also a strong reduction of cancer cell adhesion and migration. Moreover we show that impaired survival of cancer cells upon FR054 treatment is associated with the activation of the Unfolded Protein Response (UPR) and accumulation of intracellular ROS. Finally, we show that FR054 suppresses cancer growth in MDA-MB-231 xenograft mice, supporting the advantage of targeting HBP for therapeutic purpose and encouraging further investigation about the use of this small molecule as a promising compound for breast cancer therapy.

The hexosamine biosynthetic pathway (HBP) culminates in the attachment of O-linked β-N-acetylglucosamine (O-GlcNAc) onto serine/threonine residues of target proteins. The HBP is regulated by several modulators, i.e. O-linked β-N-acetylglucosaminyl transferase (OGT) and β-N-acetylglucosaminidase (OGA) catalyze the addition and removal of O-GlcNAc moieties, respectively; while flux is controlled by the rate-limiting enzyme glutamine:fructose-6-phosphate amidotransferase (GFPT), transcribed by two genes, GFPT1 and GFPT2. Since increased HBP flux is glucose-responsive and linked to insulin resistance/type 2 diabetes onset, we hypothesized that diabetic individuals exhibit differential expression of HBP regulatory genes. Volunteers (n = 60; n = 20 Mixed Ancestry, n = 40 Caucasian) were recruited from Stellenbosch and Paarl (Western Cape, South Africa) and classified as control, pre- or diabetic according to fasting plasma glucose and HbA1c levels, respectively. RNA was purified from leukocytes isolated from collected blood samples and OGT, OGA, GFPT1 and GFPT2 expressions determined by quantitative real-time PCR. The data reveal lower OGA expression in diabetic individuals (P

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

Minor amounts of glucose incorporated into the cells is metabolized via the hexosamine biosynthetic pathway, resulting in the production of uridine-5’-diphosphate-N-acetylglucosamine (UDP-GlcNAc), which is utilized as a donor substrate for the N- and O-linked glycosylation of extracellular and membrane proteins in the Golgi apparatus, or for the O-linked GlcNAc modification (O-GlcNAcylation) of intracellular proteins in the cytosol. In particular, O-GlcNAcylation, the addition of GlcNAc to serine/threonine residues, is reversibly regulated by the enzymatic activities of O-GlcNAc transferase (OGT) and O-GlcNAcase. Since OGT activity is sensitive to the intracellular UDP-GlcNAc level, flux via the hexosamine pathway influences O-GlcNAcylation. Actually, excess flux via the hexosamine pathway and resultant increased O-GlcNAcylation are associated with several pathophysiological conditions, including insulin resistance. To date, there are several studies addressing the effects of exercise on the hexosamine pathway and O-GlcNAcylation. Thus, this short review focuses on the relationships among exercise, the hexosamine pathway, O-GlcNAcylation, and insulin resistance.

Increased O-GlcNAcylation is emerging as a general characteristic of cancer cells that is critical for multiple oncogenic phenotypes. Recently, we demonstrated that elevated O-GlcNAcylation contributes to the metabolic shift seen in cancer through stabilization of the glycolytic regulator HIF-1α and links metabolism to stress and cancer cell survival.

Increased flux through the hexosamine biosynthetic pathway with glutamine:fructose-6-phosphate-amidotransferase (GFAT) as rate-limiting enzyme has been linked to the enhanced bioactivity of the prosclerotic cytokine transforming growth factor beta1 (TGF-beta1) in fibrotic complications, particularly in diabetic kidney disease. Here, we investigate in a stable transfection system the effect of overexpression of GFAT on TGF-beta1 synthesis in NIH-3T3 fibroblasts. We demonstrate a 1.8-fold stimulation of TGF-beta1 mRNA and a 1.9-fold increased protein expression, whereas TGF-beta2 production was not upregulated. The 1.5-fold enhanced TGF-beta1 promoter activity suggests a transcriptional regulation. The elevated TGF-beta1 protein is biologically active since GFAT-overexpressing cells exhibit a 2-fold fibronectin production. The results indicate a GFAT-dependent induction of TGF-beta1 synthesis.