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Purpose This phase I trial was initiated to evaluate the safety, pharmacokinetics (PK) and maximum tolerated dose (MTD) of the glycolytic inhibitor, 2-deoxy- d -glucose (2DG) in combination with docetaxel, in patients with advanced solid tumors. Methods A modified accelerated titration design was used. 2DG was administered orally once daily for 7 days every other week starting at a dose of 2 mg/kg and docetaxel was administered intravenously at 30 mg/m 2 for 3 of every 4 weeks beginning on day 1 of week 2. Following the completion of dose escalation, cohorts of patients were then treated with 2DG for 21 days or every day of each 4-week cycle for up to 12 cycles. Results Thirty-four patients were enrolled: 21 on every other week, 6 on a 21 of 28-day cycle and 7 on the continuous 2DG dosing schedule. There were no dose-limiting toxicities which met the MTD criteria. The most common adverse events were fatigue, sweating, dizziness and nausea mimicking the hypoglycemic symptoms expected from 2DG administration. Therefore, 63 mg/kg was selected as the clinically tolerable dose. The most significant adverse effects noted at 63–88 mg/kg doses were reversible hyperglycemia (100 %), gastrointestinal bleeding (6 %) and reversible grade 3 QTc prolongation (22 %). Eleven patients (32 %) had stable disease, 1 patient (3 %) partial response and 22 patients (66 %) progressive disease as their best response. There was no PK interaction between 2DG and docetaxel. Conclusion The recommended dose of 2DG in combination with weekly docetaxel is 63 mg/kg/day with tolerable adverse effects.

Background. Cardiac dysfunction is present in >40% of sepsis patients and is associated with mortality rates of up to 70%. Recent evidence suggests that glycolytic metabolism plays a critical role in host defense and inflammation. Activation of Toll-like receptors on immune cells can enhance glycolytic metabolism. This study investigated whether modulation of glycolysis by inhibition of hexokinase will be beneficial to septic cardiomyopathy. Methods. Male C57B6/J mice were treated with a hexokinase inhibitor (2-deoxy-D-glucose [2-DG], 0.25–2 g/kg, n = 6–8) before cecal ligation and puncture (CLP) induced sepsis. Untreated septic mice served as control. Sham surgically operated mice treated with or without the 2-DG inhibitor served as sham controls. Cardiac function was assessed 6 hours after CLP sepsis by echocardiography. Serum was harvested for measurement of inflammatory cytokines and lactate. Results. Sepsis-induced cardiac dysfunction was significantly attenuated by administration of 2-DG. Ejection fraction and fractional shortening in 2-DG–treated septic mice were significantly (P < .05) greater than in untreated CLP mice. 2-DG administration also significantly improved survival outcome, reduced kidney and liver injury, attenuated sepsis-increased serum levels of tumor necrosis factor α and interleukin 1β as well as lactate, and enhanced the expression of Sirt1 and Sirt3 in the myocardium, which play an important role in mitochondrial function and metabolism. In addition, 2-DG administration suppresses sepsis-increased expression of apoptotic inducers Bak and Bax as well as JNK phosphorylation in the myocardium. Conclusions. Glycolytic metabolism plays an important role in mediating sepsis-induced septic cardiomyopathy. The mechanisms may involve regulation of inflammatory response and apoptotic signaling.

Nanocatalytic immunotherapy holds excellent potential for future cancer therapy due to its rapid activation of the immune system to attack tumor cells. However, a high level of N-glycosylation can protect tumor cells, compromising the anticancer immunity of nanocatalytic immunotherapy. Here, we show a 2-deoxyglucose (2-DG) and bismuth ferrite co-loaded gel (DBG) scaffold for enhanced cancer piezocatalytic immunotherapy. After the implantation in the tumor, DBG generates both reactive oxygen species (ROS) and piezoelectric signals when excited with ultrasound irradiation, significantly promoting the activation of anticancer immunity. Meanwhile, 2-DG released from ROS-sensitive DBG disrupts the N-glycans synthesis, further overcoming the immunosuppressive microenvironment of tumors. The synergy effects of ultrasound-triggered and glycosylation inhibition enhanced tumor piezocatalytic immunotherapy are demonstrated on four mouse cancer models. A “hot” tumor-immunity niche is produced to inhibit tumor progress and lung metastasis and elicit strong immune memory effects. This work provides a promising piezocatalytic immunotherapy for malignant solid tumors featuring both low immunogenicity and high levels of N-glycosylation. N-glycosylation has a significant role in immunosuppressive tumor microenvironment (TME). Here, the authors design an injectable ROS-sensitive hydrogel scaffold co-delivering piezocatalyst BiFeO 3 and glucose/mannose analog 2-deoxy-d-glucose alleviating N-glycosylation inhibition and thereby effectively stimulating immune responses within TME.

Uptake of administered D-glucose (Glc) or 2-deoxy-D-glucose (2DG) has been indirectly mapped through the chemical exchange (CE) between glucose hydroxyl and water protons using CE-dependent saturation transfer (glucoCEST) magnetic resonance imaging (MRI). We propose an alternative technique—on-resonance CE-sensitive spin-lock (CESL) MRI—to enhance responses to glucose changes. Phantom data and simulations suggest higher sensitivity for this ‘glucoCESL’ technique (versus glucoCEST) in the intermediate CE regime relevant to glucose. Simulations of CESL signals also show insensitivity to B0-fluctuations. Several findings are apparent from in vivo glucoCESL studies of rat brain at 9.4 Tesla with intravenous injections. First, dose-dependent responses are nearly linearly for 0.25-, 0.5-, and 1-g/kg Glc administration (obtained with 12-second temporal resolution), with changes robustly detected for all doses. Second, responses at a matched dose of 1 g/kg are much larger and persist for a longer duration for 2DG versus Glc administration, and are minimal for mannitol as an osmolality control. And third, with similar increases in steady-state blood glucose levels, glucoCESL responses are ~2.2 times higher for 2DG versus Glc, consistent with their different metabolic properties. Overall, we show that glucoCESL MRI could be a highly sensitive and quantifiable tool for glucose transport and metabolism studies.

ZBTB7A is frequently mutated in acute myeloid leukemia (AML) with t(8;21) translocation. However, the oncogenic collaboration between mutated ZBTB7A and the RUNX1–RUNX1T1 fusion gene in AML t(8;21) remains unclear. Here, we investigate the role of ZBTB7A and its mutations in the context of normal and malignant hematopoiesis. We demonstrate that clinically relevant ZBTB7A mutations in AML t(8;21) lead to loss of function and result in perturbed myeloid differentiation with block of the granulocytic lineage in favor of monocytic commitment. In addition, loss of ZBTB7A increases glycolysis and hence sensitizes leukemic blasts to metabolic inhibition with 2-deoxy-d-glucose. We observed that ectopic expression of wild-type ZBTB7A prevents RUNX1-RUNX1T1-mediated clonal expansion of human CD34+ cells, whereas the outgrowth of progenitors is enabled by ZBTB7A mutation. Finally, ZBTB7A expression in t(8;21) cells lead to a cell cycle arrest that could be mimicked by inhibition of glycolysis. Our findings suggest that loss of ZBTB7A may facilitate the onset of AML t(8;21), and that RUNX1-RUNX1T1-rearranged leukemia might be treated with glycolytic inhibitors.

Glucose transporters are essential for metabolism of glucose in cells of diverse organisms from microbes to humans, exemplified by the disease-related human proteins GLUT1, 2, 3 and 4. Despite rigorous efforts, the structural information for GLUT1–4 or their homologues remains largely unknown. Here we report three related crystal structures of XylE, an Escherichia coli homologue of GLUT1–4, in complex with d -xylose, d -glucose and 6-bromo-6-deoxy- d -glucose, at resolutions of 2.8, 2.9 and 2.6 Å, respectively. The structure consists of a typical major facilitator superfamily fold of 12 transmembrane segments and a unique intracellular four-helix domain. XylE was captured in an outward-facing, partly occluded conformation. Most of the important amino acids responsible for recognition of d -xylose or d -glucose are invariant in GLUT1–4, suggesting functional and mechanistic conservations. Structure-based modelling of GLUT1–4 allows mapping and interpretation of disease-related mutations. The structural and biochemical information reported here constitutes an important framework for mechanistic understanding of glucose transporters and sugar porters in general. A study of X-ray crystal structures of the Escherichia coli xylose transporter XylE, which is a bacterial homologue of the human glucose transporters GLUT1–4, complexed with glucose and its analogues yields a framework for understanding the molecular mechanism by which membrane proteins transport glucose and other sugars across cell membranes. Glucose transporter structure determined Proteins that transport glucose across cellular membranes are essential for glucose metabolism in many organisms, from microbes to mammals. This Article reports three X-ray crystal structures of XylE — an Escherichia coli homologue of the GLUT family of human proteins — in complex with D -xylose, D -glucose and 6-bromo-6-deoxy- D -glucose. Structure-based modelling of GLUT1–4 enabled the authors to map known disease-related mutations, and the structural and biochemical information reported here provides a framework for understanding the molecular mechanism by which membrane proteins transport glucose and other sugars.

5-Aminoimidazole-4-Carboxamide 1-β- d -Ribofuranoside Acutely Stimulates Skeletal Muscle 2-Deoxyglucose Uptake in Healthy Men Daniel J. Cuthbertson 1 , John A. Babraj 2 , Kirsteen J.W. Mustard 3 , Mhairi C. Towler 4 , Kevin A. Green 3 , Henning Wackerhage 4 , Graeme P. Leese 1 , Keith Baar 3 , Michaela Thomason-Hughes 3 , Calum Sutherland 5 , D. Grahame Hardie 3 and Michael J. Rennie 6 1 Department of Medicine, Ninewells Hospital and Medical School, University of Dundee, Dundee, Scotland 2 School of Life Sciences, Heriot Watt University, Edinburgh, Scotland 3 Division of Molecular Physiology, College of Life Sciences, University of Dundee, Dundee, Scotland 4 College of Life Sciences and Medicine, University of Aberdeen, Aberdeen, Scotland 5 Department of Pharmacology and Neurosciences, University of Dundee, Dundee, Scotland 6 School of Biomedical Sciences, Graduate Entry Medical School, University of Nottingham, Derby City Hospital, Derby, U.K Address correspondence and reprint requests to Dr. Daniel Cuthbertson, Department of Medicine, Ninewells Hospital and Medical School, Dundee, Scotland DD1 9SY. E-mail: d.j.r.cuthbertson{at}dundee.ac.uk Abstract Activation of AMP-activated protein kinase (AMPK) in rodent muscle by exercise, metformin, 5-aminoimidazole-4-carboxamide 1-β- d -ribofuranoside (AICAR), and adiponectin increases glucose uptake. The aim of this study was to determine whether AICAR stimulates muscle glucose uptake in humans. We studied 29 healthy men (aged 26 ± 8 years, BMI 25 ± 4 kg/m 2 [mean ± SD]). Rates of muscle 2-deoxyglucose (2DG) uptake were determined by measuring accumulation of total muscle 2DG (2DG and 2DG-6-phosphate) during a primed, continuous 2DG infusion. The effects of AICAR and exercise on muscle AMPK activity/phosphorylation and 2DG uptake were determined. Whole-body glucose disposal was compared before and during AICAR with the euglycemic-hyperinsulinemic clamp. Muscle 2DG uptake was linear over 9 h ( R 2 = 0.88 ± 0.09). After 3 h, 2DG uptake increased 2.1 ± 0.8- and 4.7 ± 1.7-fold in response to AICAR or bicycle exercise, respectively. AMPK α 1 and α 2 activity or AMPK phosphorylation was unchanged after 20 min or 3 h of AICAR, but AMPK phosphorylation significantly increased immediately and 3 h after bicycle exercise. AICAR significantly increased phosphorylation of extracellular signal–regulated kinase 1/2, but phosphorylation of β-acetyl-CoA carboxylase, glycogen synthase, and protein kinase B or insulin receptor substrate-1 level was unchanged. Mean whole-body glucose disposal increased by 7% with AICAR from 9.3 ± 0.6 to 10 ± 0.6 mg · kg −1 · min −1 ( P < 0.05). In healthy people, AICAR acutely stimulates muscle 2DG uptake with a minor effect on whole-body glucose disposal. 2DG, 2-deoxyglucose 2DG6P, 2-deoxyglucose 6-phosphate ACC, acetyl-CoA carboxylase AICAR, 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside AMPK, AMP-activated protein kinase aPKC, atypical protein kinase C ERK, extracellular signal–regulated kinase FDG, fluorodeoxyglucose GS, glycogen synthase GSK, glycogen synthase kinase IRS-1, insulin receptor substrate-1 PKB, protein kinase B ZMP, 5-aminoimidazole-4-carboxamide ribonucleoside Footnotes Published ahead of print at http://diabetes.diabetesjournals.org on 18 May 2007. DOI: 10.2337/db06-1716. D.J.C. and J.A.B. contributed equally to this work. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Accepted May 11, 2007. Received December 8, 2006. DIABETES

The amino acid and oligopeptide transporter Solute carrier family 15 member A4 (SLC15A4), which resides in lysosomes and is preferentially expressed in immune cells, plays critical roles in the pathogenesis of lupus and colitis in murine models. Toll-like receptor (TLR) 7/9- and nucleotide-binding oligomerization domain-containing protein 1 (NOD1)-mediated inflammatory responses require SLC15A4 function for regulating themechanistic target of rapamycin complex 1 (mTORC1) or transporting L-Ala-γ-D-Glu-meso-diaminopimelic acid, IL-12: interleukin-12 (Tri-DAP), respectively. Here, we further investigated the mechanism of how SLC15A4 directs inflammatory responses. Proximity-dependent biotin identification revealed glycolysis as highly enriched gene ontology terms. Fluxome analyses in macrophages indicated that SLC15A4 loss causes insufficient biotransformation of pyruvate to the tricarboxylic acid cycle, while increasing glutaminolysis to the cycle. Furthermore, SLC15A4 was required for M1-prone metabolic change and inflammatory IL-12 cytokine productions after TLR9 stimulation. SLC15A4 could be in close proximity to AMP-activated protein kinase (AMPK) and mTOR, and SLC15A4 deficiency impaired TLR-mediated AMPK activation. Interestingly, SLC15A4-intact but not SLC15A4-deficient macrophages became resistant to fluctuations in environmental nutrient levels by limiting the use of the glutamine source; thus, SLC15A4 was critical for macrophage’s respiratory homeostasis. Our findings reveal a mechanism of metabolic regulation in which an amino acid transporter acts as a gatekeeper that protects immune cells’ ability to acquire an M1-prone metabolic phenotype in inflammatory tissues by mitigating metabolic stress.

The glucose analogue 2-deoxyglucose (2DG) has frequently been used as a tool to study cellular glucose uptake and to inhibit glycolysis. Exposure of primary cultured astrocytes to 2DG caused a time- and concentration-dependent cellular accumulation of 2-deoxyglucose-6-phosphate (2DG6P) that was accompanied by a rapid initial decline in cellular ATP content. Inhibitors of mitochondrial respiration as well as inhibitors of mitochondrial uptake of pyruvate and activated fatty acids accelerated the ATP loss, demonstrating that mitochondrial ATP regeneration contributes to the partial maintenance of the ATP content in 2DG-treated astrocytes. After a 30 min exposure to 10 mM 2DG the specific content of cellular 2DG6P had accumulated to around 150 nmol/mg, while cellular ATP was lowered by 50% to around 16 nmol/mg. Following such a 2DG6P-loading of astrocytes, glycolytic lactate production from applied glucose was severely impaired during the initial 60 min of incubation, but was reestablished during longer incubation concomitant with a loss in cellular 2DG6P content. In contrast to glycolysis, the glucose-dependent NADPH regeneration via the pentose phosphate pathway (PPP) was only weakly affected in 2DG6P-loaded astrocytes and in cells that were coincubated with glucose in the presence of an excess of 2DG. Additionally, in the presence of 2DG PPP-dependent WST1 reduction was found to have doubled compared to hexose-free control incubations, indicating that cellular 2DG6P can serve as substrate for NADPH regeneration by the astrocytic PPP. The data presented provide new insights on the metabolic consequences of a 2DG exposure on the energy and glucose metabolism of astrocytes and demonstrate the reversibility of the inhibitory potential of a 2DG-treatment on the glucose metabolism of cultured astrocytes.

Bacillus anthracis , a spore-forming gram-positive bacterium, causes anthrax. The external surface of the exosporium is coated with glycosylated proteins. The sugar additions are capped with the unique monosaccharide anthrose. The West African Group (WAG) B . anthracis have mutations rendering them anthrose deficient. Through genome sequencing, we identified 2 different large chromosomal deletions within the anthrose biosynthetic operon of B . anthracis strains from Chile and Poland. In silico analysis identified an anthrose-deficient strain in the anthrax outbreak among European heroin users. Anthrose-deficient strains are no longer restricted to West Africa so the role of anthrose in physiology and pathogenesis was investigated in B . anthracis Sterne. Loss of anthrose delayed spore germination and enhanced sporulation. Spores without anthrose were phagocytized at higher rates than spores with anthrose, indicating that anthrose may serve an antiphagocytic function on the spore surface. The anthrose mutant had half the LD 50 and decreased time to death (TTD) of wild type and complement B . anthracis Sterne in the A/J mouse model. Following infection, anthrose mutant bacteria were more abundant in the spleen, indicating enhanced dissemination of Sterne anthrose mutant. At low sample sizes in the A/J mouse model, the mortality of Δ antC -infected mice challenged by intranasal or subcutaneous routes was 20% greater than wild type. Competitive index (CI) studies indicated that spores without anthrose disseminated to organs more extensively than a complemented mutant. Death process modeling using mouse mortality dynamics suggested that larger sample sizes would lead to significantly higher deaths in anthrose-negative infected animals. The model was tested by infecting Galleria mellonella with spores and confirmed the anthrose mutant was significantly more lethal. Vaccination studies in the A/J mouse model showed that the human vaccine protected against high-dose challenges of the nonencapsulated Sterne-based anthrose mutant. This work begins to identify the physiologic and pathogenic consequences of convergent anthrose mutations in B . anthracis .