CD205+ polymorphonuclear myeloid-derived suppressor cells suppress antitumor immunity by overexpressing GLUT3.

Myeloid-derived suppressor cells (MDSCs) are responsible for antitumor immunodeficiency in tumor-bearing hosts. Primarily, MDSCs are classified into 2 groups: monocytic (M)-MDSCs and polymorphonuclear (PMN)-MDSCs. In most cancers, PMN-MDSCs (CD11b+ Ly6Clow Ly6G+ cells) represent the most abundant MDSC subpopulation. However, the functional and phenotypic heterogeneities of PMN-MDSC remain elusive, which delays clinical therapeutic targeting decisions. In the 4T1 murine tumor model, CD11b+ Ly6Glow PMN-MDSCs were sensitive to surgical and pharmacological interventions. By comprehensively analyzing 64 myeloid cell-related surface molecule expression profiles, cell density, nuclear morphology, and immunosuppressive activity, the PMN-MDSC population was further classified as CD11b+ Ly6Glow CD205+ and CD11b+ Ly6Ghigh TLR2+ subpopulations. The dichotomy of PMN-MDSCs based on CD205 and TLR2 is observed in 4T07 murine tumor models (but not in EMT6). Furthermore, CD11b+ Ly6Glow CD205+ cells massively accumulated at the spleen and liver of tumor-bearing mice, and their abundance correlated with in situ tumor burdens (with or without intervention). Moreover, we demonstrated that CD11b+ Ly6Glow CD205+ cells were sensitive to glucose deficiency and 2-deoxy-d-glucose (2DG) treatment. Glucose transporter 3 (GLUT3) knockdown by siRNA significantly triggered apoptosis and reduced glucose uptake in CD11b+ Ly6Glow CD205+ cells, demonstrating the dependence of CD205+ PMN-MDSCs survival on both glucose uptake and GLUT3 overexpression. As GLUT3 has been recognized as a target for the rescue of host antitumor immunity, our results further directed the PMN-MDSC subsets into the CD205+ GLUT3+ subpopulation as future targeting therapy.

Small-Molecule Inhibition of Glucose Transporters GLUT-1-4.

Glucose addiction is observed in cancer and other diseases that are associated with hyperproliferation. The development of compounds that restrict glucose supply and decrease glycolysis has great potential for the development of new therapeutic approaches. Addressing facilitative glucose transporters (GLUTs), which are often upregulated in glucose-dependent cells, is therefore of particular interest. This article reviews a selection of potent, isoform-selective GLUT inhibitors and their biological characterization. Potential therapeutic applications of GLUT inhibitors in oncology and other diseases that are linked to glucose addiction are discussed.

Synthesis of Indomorphan Pseudo-Natural Product Inhibitors of Glucose Transporters GLUT-1 and -3.

Bioactive compound design based on natural product (NP) structure may be limited because of partial coverage of NP-like chemical space and biological target space. These limitations can be overcome by combining NP-centered strategies with fragment-based compound design through combination of NP-derived fragments to afford structurally unprecedented "pseudo-natural products" (pseudo-NPs). The design, synthesis, and biological evaluation of a collection of indomorphan pseudo-NPs that combine biosynthetically unrelated indole- and morphan-alkaloid fragments are described. Indomorphane derivative Glupin was identified as a potent inhibitor of glucose uptake by selectively targeting and upregulating glucose transporters GLUT-1 and GLUT-3. Glupin suppresses glycolysis, reduces the levels of glucose-derived metabolites, and attenuates the growth of various cancer cell lines. Our findings underscore the importance of dual GLUT-1 and GLUT-3 inhibition to efficiently suppress tumor cell growth and the cellular rescue mechanism, which counteracts glucose scarcity.

Dynamic Profiling of Insulin Secretion and ATP Generation in Isolated Human and Mouse Islets Reveals Differential Glucose Sensitivity.

BACKGROUND/AIMS: Rodent islets are often used for basic science research but they do not always recapitulate signalling events in human islets. This study evaluated the glucose-dependent responses of human and mouse islets in terms of dynamic insulin secretion, metabolic coupling and the role of glucose transporters. METHODS: Glucose-induced insulin secretion from isolated mouse and human islets was profiled by perifusion and islet ATP levels were measured by chemoluminescence assay. Glucose transporter expression was determined by qPCR and western blotting. RESULTS: Human islets show a left-shifted glucose concentration-insulin secretion profile compared to mouse islets. These data are consistent with glucose transporter expression, with human islets expressing mainly GLUT1 and GLUT3, and GLUT2 being the predominant transporter in mouse islets. Using the GLUT1 inhibitor STF-31 we unveiled an important role for GLUT1 for differences in glucose-induced insulin secretion profiles observed between the two species. CONCLUSION: The high affinity of GLUT1/3 for glucose reflects the left-shifted glucose-induced insulin secretory response of human islets and the impairment of insulin secretion from human islets after STF-31 treatment indicates an important role for GLUT1 in human islet stimulus-secretion coupling. Our data provide further insight into key differences between insulin secretion regulation in mouse and human islets.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

Ascorbic acid-dependent GLUT3 inhibition is a critical step for switching neuronal metabolism.

Intracellular ascorbic acid is able to modulate neuronal glucose utilization between resting and activity periods. We have previously demonstrated that intracellular ascorbic acid inhibits deoxyglucose transport in primary cultures of cortical and hippocampal neurons and in HEK293 cells. The same effect was not seen in astrocytes. Since this observation was valid only for cells expressing glucose transporter 3 (GLUT3), we evaluated the importance of this transporter on the inhibitory effect of ascorbic acid on glucose transport. Intracellular ascorbic acid was able to inhibit (3)H-deoxyglucose transport only in astrocytes expressing GLUT3-EGFP. In C6 glioma cells and primary cultures of cortical neurons, which natively express GLUT3, the same inhibitory effect on (3)H-deoxyglucose transport and fluorescent hexose 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2-NBDG) was observed. Finally, knocking down the native expression of GLUT3 in primary cultured neurons and C6 cells using shRNA was sufficient to abolish the ascorbic acid-dependent inhibitory effect on uptake of glucose analogs. Uptake assays using real-time confocal microscopy demonstrated that ascorbic acid effect abrogation on 2-NBDG uptake in cultured neurons. Therefore, ascorbic acid would seem to function as a metabolic switch inhibiting glucose transport in neurons under glutamatergic synaptic activity through direct or indirect inhibition of GLUT3.

Cystine transporter regulation of pentose phosphate pathway dependency and disulfide stress exposes a targetable metabolic vulnerability in cancer.

SLC7A11-mediated cystine uptake is critical for maintaining redox balance and cell survival. Here we show that this comes at a significant cost for cancer cells with high levels of SLC7A11. Actively importing cystine is potentially toxic due to its low solubility, forcing cancer cells with high levels of SLC7A11 (SLC7A11high) to constitutively reduce cystine to the more soluble cysteine. This presents a significant drain on the cellular NADPH pool and renders such cells dependent on the pentose phosphate pathway. Limiting glucose supply to SLC7A11high cancer cells results in marked accumulation of intracellular cystine, redox system collapse and rapid cell death, which can be rescued by treatments that prevent disulfide accumulation. We further show that inhibitors of glucose transporters selectively kill SLC7A11high cancer cells and suppress SLC7A11high tumour growth. Our results identify a coupling between SLC7A11-associated cystine metabolism and the pentose phosphate pathway, and uncover an accompanying metabolic vulnerability for therapeutic targeting in SLC7A11high cancers.

GLUT1 and GLUT3 involvement in anthocyanin gastric transport- Nanobased targeted approach.

Anthocyanins may protect against a myriad of human diseases. However few studies have been conducted to evaluate their bioavailability so their absorption mechanism remains unclear. This study aimed to evaluate the role of two glucose transporters (GLUT1 and GLUT3) in anthocyanins absorption in the human gastric epithelial cells (MKN-28) by using gold nanoparticles to silence these transporters. Anthocyanins were purified from purple fleshed sweet potatoes and grape skin. Silencing of GLUT1 and/or GLUT3 mRNA was performed by adding AuNP@GLUT1 and/or AuNP@GLUT3 to MKN-28 cells. Downregulation of mRNA expression occurred concomitantly with the reduction in protein expression. Malvidin-3-O-glucoside (Mv3glc) transport was reduced in the presence of either AuNP@GLUT1 and AuNP@GLUT3, and when both transporters were blocked simultaneously. Peonidin-3-(6'-hydroxybenzoyl)-sophoroside-5-glucoside (Pn3HBsoph5glc) and Peonidin-3-(6'-hydroxybenzoyl-6″-caffeoyl)-sophoroside-5-glucoside (Pn3HBCsoph5glc) were assayed to verify the effect of the sugar moiety esterification at glucose B in transporter binding. Both pigments were transported with a lower transport efficiency compared to Mv3glc, probably due to steric hindrance of the more complex structures. Interestingly, for Pn3HBCsoph5glc although the only free glucose is at C5 and the inhibitory effect of the nanoparticles was also observed, reinforcing the importance of glucose on the transport regardless of its position or substitution pattern. The results support the involvement of GLUT1 and GLUT3 in the gastric absorption of anthocyanins.

Inhibition of Glucose Transporters and Glutaminase Synergistically Impairs Tumor Cell Growth.

Cancer cells sustain growth by altering their metabolism to accelerated aerobic glycolysis accompanied by increased glucose demand and employ glutamine as additional nutrient source. This metabolic adaptation induces upregulation of glucose transporters GLUT-1 and -3, and simultaneous targeting of both transporters and of glutamine metabolism may offer a promising approach to inhibit cancer cell growth. We describe the discovery of the very potent glucose uptake inhibitor Glutor, which targets glucose transporters GLUT-1, -2, and -3, attenuates glycolytic flux and potently and selectively suppresses growth of a variety of cancer cell lines. Co-treatment of colon cancer cells with Glutor and glutaminase inhibitor CB-839 very potently and synergistically inhibits cancer cell growth. Such a dual inhibition promises to be particularly effective because it targets the metabolic plasticity as well as metabolic rescue mechanisms in cancer cells.

Metastatic Colorectal Cancer Rewrites Metabolic Program Through a Glut3-YAP-dependent Signaling Circuit.

Rationale: Cancer cells reprogram cellular metabolism to fulfill their needs for rapid growth and metastasis. However, the mechanism controlling this reprogramming is poorly understood. We searched for upregulated signaling in metastatic colorectal cancer and investigated the mechanism by which Glut3 promotes tumor metastasis. Methods: We compared RNA levels and glycolytic capacity in primary and metastatic colon cancer. The expression and association of Glut3 with clinical prognosis in colon cancer tissues was determined by immunohistochemistry. Glut3 gain-of-function and loss-of-function were established using colon cancer HCT116, HT29, and metastatic 116-LM cells, and tumor invasiveness and stemness properties were evaluated. Metabolomic profiles were analyzed by GC/MS and CE-TOF/MS. The metastatic burden in mice fed a high-fat sucrose diet was assessed by intravenous inoculation with Glut3 knockdown 116-LM cells. Results: Upregulation of glycolytic genes and glycolytic capacity was detected in metastatic colorectal cancer cells. Specifically, Glut3 overexpression was associated with metastasis and poor survival in colorectal cancer patients. Mechanistically, Glut3 promoted invasiveness and stemness in a Yes-associated protein (YAP)-dependent manner. Activation of YAP in turn transactivated Glut3 and regulated a group of glycolytic genes. Interestingly, the expression and phosphorylation of PKM2 were concomitantly upregulated in metastatic colorectal cancer, and it was found to interact with YAP and enhance the expression of Glut3. Importantly, a high-fat high-sucrose diet promoted tumor metastasis, whereas the inhibition of either Glut3 or YAP effectively reduced the metastatic burden. Conclusion: Activation of the Glut3-YAP signaling pathway acts as a master activator to reprogram cancer metabolism and thereby promotes metastasis. Our findings reveal the importance of metabolic reprogramming in supporting cancer metastasis as well as possible therapeutic targets.

The effect of PPAR-γ agonist on (18)F-FDG PET imaging for differentiating tumors and inflammation lesions.

INTRODUCTION: (18)F-2-deoxy-2-fluoro-d-glucose ((18)F-FDG) positron emission tomography (PET) has been used for imaging human cancers for several decades. Despite its extensive use, (18)F-FDG PET imaging has limitations in the tumor findings. The goal of this study was to investigate the potential of a PPAR-γ agonist pioglitazone (PIO) to distinguish tumors and inflammatory lesions in (18)F-FDG PET imaging. METHODS: Studies of cellular uptake of (18)F-FDG and Western blot were performed in macrophage (RAW264.7) and three tumor cell lines (A549, KB, and MDA-MB-231) after treatment with PIO. In vivo microPET/CT imaging and biodistribution were performed in animal models. RESULTS: The uptake of (18)F-FDG in the macrophages was decreased and uptake of (18)F-FDG in the tumor cells was increased when these cells were treated with PIO. Western blot showed that the expression of Glut1 was reduced by treatment of PIO in the macrophage cells, whereas the expression of Glut1 in the tumor cells was increased. In vivo PET/CT imaging revealed that (18)F-FDG uptake (%ID/g) in the tumors was enhanced from 4.05±1.46 to 5.28±1.92 for A549, from 3.9±0.5 to 4.9±0.2 for KB, and from 9.14±0.86 to 13.48±2.07 for MDA-MB-231 tumors after treatment with PIO. Unlike tumors, the RAW264.7 xenograft model showed the reduced (18)F-FDG uptake in the inflammatory lesion from 11.74±1.19 to 6.50±1.47. The results of biodistribution also showed that (18)F-FDG uptake in the tumors were increased after treatment of PIO. However, the uptake of inflammation lesions was reduced. CONCLUSIONS: In this study, we demonstrated the effect of a PPAR-γ agonist PIO on (18)F-FDG uptake in tumors and inflammation in vitro and in vivo. PIO has potential to differentiate tumors and inflammatory lesions on (18)F-FDG PET imaging.

Osteopontin upregulates the expression of glucose transporters in osteosarcoma cells.

Osteosarcoma is the most common primary malignancy of bone. Even after the traditional standard surgical therapy, metastasis still occurs in a high percentage of patients. Glucose is an important source of metabolic energy for tumor proliferation and survival. Tumors usually overexpress glucose transporters, especially hypoxia-responsive glucose transporter 1 and glucose transporter 3. Osteopontin, hypoxia-responsive glucose transporter 1, and glucose transporter 3 are overexpressed in many types of tumors and have been linked to tumorigenesis and metastasis. In this study, we investigated the regulation of glucose transporters by osteopontin in osteosarcoma. We observed that both glucose transporters and osteopontin were upregulated in hypoxic human osteosarcoma cells. Endogenously released osteopontin regulated the expression of glucose transporter 1 and glucose transporter 3 in osteosarcoma and enhanced glucose uptake into cells via the αvß3 integrin. Knockdown of osteopontin induced cell death in 20% of osteosarcoma cells. Phloretin, a glucose transporter inhibitor, also caused cell death by treatment alone. The phloretin-induced cell death was significantly enhanced in osteopontin knockdown osteosarcoma cells. Combination of a low dose of phloretin and chemotherapeutic drugs, such as daunomycin, 5-Fu, etoposide, and methotrexate, exhibited synergistic cytotoxic effects in three osteosarcoma cell lines. Inhibition of glucose transporters markedly potentiated the apoptotic sensitivity of chemotherapeutic drugs in osteosarcoma. These results indicate that the combination of a low dose of a glucose transporter inhibitor with cytotoxic drugs may be beneficial for treating osteosarcoma patients.

Does the association with diabetes say more about schizophrenia and its treatment?--the GLUT hypothesis.

All effective anti psychotic drugs block glucose transporter proteins (GLUTs), peripherally and in the brain. These drugs are implicated in hyperglycaemia as demonstrated in mouse and human studies. Clozapine is the strongest blocker, with Haloperidol the weakest. The GLUT hypothesis suggests that schizophrenia is partly due to poor functioning of brain glucose transporters (GLUT 1 and 3). Neuronal glucose malnutrition could result in excessive neuronal pruning (so called Crow's Type 2 with a predominance of negative symptoms) or result in recurrent/ineffective pruning (Type 1 with positive symptoms). GLUT blockade by anti psychotic agents could assist Type 1 patients to complete the pruning process by deactivating already damaged neurones and circuits, but will make Type 2 patients more cognitively impaired. Future treatment options are discussed in line with the above formulation.

DNA damage-induced modulation of GLUT3 expression is mediated through p53-independent extracellular signal-regulated kinase signaling in HeLa cells.

Many cancer cells exhibit increased rates of uptake and metabolism of glucose compared with normal cells. Glucose uptake in mammalian cells is mediated by the glucose transporter (GLUT) family. Here, we report that DNA-damaging anticancer agents such as Adriamycin and etoposide suppressed the expression of GLUT3, but not GLUT1, in HeLa cells and a tumorigenic HeLa cell hybrid. Suppression of GLUT3 expression determined by the real-time PCR was also evident with another DNA-damaging agent, camptothecin, which reduced the promoter's activity as determined with a luciferase-linked assay. The suppression by these agents seemed to be induced independently of p53, and it was evident when wild-type p53 was overproduced in these cells. In contrast, the mitogen-activated protein kinase/extracellular signal regulated kinase (MAPK/ERK) kinase (MEK) inhibitor U0126 (but not the phosphoinositide 3-kinase inhibitor LY294002) prevented the drug-induced suppression as determined by reverse transcription-PCR and promoter assays. Furthermore, overexpression of GLUT3 in HeLa cell hybrids increased resistance to these drugs, whereas depletion of the gene by small interfering RNA rendered the cells more sensitive to the drugs, decreasing glucose consumption. The results suggest that DNA-damaging agents reduce GLUT3 expression in cancer cells through activation of the MEK-ERK pathway independently of p53, leading to cell death or apoptosis. The findings may contribute to the development of new chemotherapeutic drugs based on the GLUT3-dependent metabolism of glucose.

Ascorbic acid participates in a general mechanism for concerted glucose transport inhibition and lactate transport stimulation.

In this paper, we present a novel function for ascorbic acid. Ascorbic acid is an important water-soluble antioxidant and cofactor in various enzyme systems. We have previously demonstrated that an increase in neuronal intracellular ascorbic acid is able to inhibit glucose transport in cortical and hippocampal neurons. Because of the presence of sodium-dependent vitamin C transporters, ascorbic acid is highly concentrated in brain, testis, lung, and adrenal glands. In this work, we explored how ascorbic acid affects glucose and lactate uptake in neuronal and non-neuronal cells. Using immunofluorescence and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis, the expression of glucose and ascorbic acid transporters in non-neuronal cells was studied. Like neurons, HEK293 cells expressed GLUT1, GLUT3, and SVCT2. With radioisotope-based methods, only intracellular ascorbic acid, but not extracellular, inhibits 2-deoxyglucose transport in HEK293 cells. As monocarboxylates such as pyruvate and lactate, are important metabolic sources, we analyzed the ascorbic acid effect on lactate transport in cultured neurons and HEK293 cells. Intracellular ascorbic acid was able to stimulate lactate transport in both cell types. Extracellular ascorbic acid did not affect this transport. Our data show that ascorbic acid inhibits glucose transport and stimulates lactate transport in neuronal and non-neuronal cells. Mammalian cells frequently present functional glucose and monocarboxylate transporters, and we describe here a general effect in which ascorbic acid functions like a glucose/monocarboxylate uptake switch in tissues expressing ascorbic acid transporters.

Role of beta-adrenoceptors in memory consolidation: beta3-adrenoceptors act on glucose uptake and beta2-adrenoceptors on glycogenolysis.

Noradrenaline, acting via beta(2)- and beta(3)-adrenoceptors (AR), enhances memory formation in single trial-discriminated avoidance learning in day-old chicks by mechanisms involving changes in metabolism of glucose and/or glycogen. Earlier studies of memory consolidation in chicks implicated beta(3)- rather than beta(2)-ARs in enhancement of memory consolidation by glucose, but did not elucidate whether stimulation of glucose uptake or of glycolysis was responsible. This study examines the role of glucose transport in memory formation using central injection of the nonselective facilitative glucose transporter (GLUT) inhibitor cytochalasin B, the endothelial/astrocytic GLUT-1 inhibitor phloretin and the Na(+)/energy-dependent endothelial glucose transporter (SGLT) inhibitor phlorizin. Cytochalasin B inhibited memory when injected into the mesopallium (avian cortex) either close to or between 25 and 45 min after training, whereas phloretin and phlorizin only inhibited memory at 30 min. This suggested that astrocytic/endothelial (GLUT-1) transport is critical at the time of consolidation, whereas a different transporter, probably the neuronal glucose transporter (GLUT-3), is important at the time of training. Inhibition of glucose transport by cytochalasin B, phloretin, or phlorizin also interfered with beta(3)-AR-mediated memory enhancement 20 min posttraining, whereas inhibition of glycogenolysis interfered with beta(2)-AR agonist enhancement of memory. We conclude that in astrocytes (1) activities of both GLUT-1 and SGLT are essential for memory consolidation 30 min posttraining; (2) neuronal GLUT-3 is essential at the time of training; and (3) beta(2)- and beta(3)-ARs consolidate memory by different mechanisms; beta(3)-ARs stimulate central glucose transport, whereas beta(2)-ARs stimulate central glycogenolysis.