Inhibition Of GLUT1 Research Articles

The significance of upper glycolytic components in regulating retinal pigment epithelial cellular behavior

Cell adhesion to the extracellular matrix and its natural outcome of cell spreading, along with the maintenance of barrier activity, are essential behaviors of epithelial cells, including retinal pigment epithelium (RPE). Disruptions in these characteristics can result in severe vision-threatening diseases such as diabetic macular edema and age-related macular degeneration. However, the precise mechanisms underlying how RPE cells regulate their barrier integrity and cell spreading are not fully understood. This study aims to elucidate the relative importance of upper glycolytic components in governing these cellular behaviors of RPE cells. Electric Cell-Substrate Impedance Sensing (ECIS) technology was utilized to assess in real-time the effects of targeting various upper glycolytic enzymes on RPE barrier function and cell spreading by measuring cell resistance and capacitance, respectively. Specific inhibitors used included WZB117 for Glut1 inhibition, Lonidamine for Hexokinase inhibition, PFK158 for PFKFB3/PFK axis inhibition, and TDZD-8 for Aldolase inhibition. Additionally, the viability of RPE cells was evaluated using a lactate dehydrogenase (LDH) cytotoxicity assay. The most significant decrease in electrical resistance and increase in capacitance of RPE cells were observed due to dose-dependent inhibition of Glut1 using WZB117, as well as Aldolase inhibition with TDZD-8. LDH level analysis at 24–72 h post-treatment with WZB117 (1 and 10 μM) or TDZD-8 (1 μM) showed no significant difference compared to the control, indicating that the disruption of RPE functionality was not attributed to cell death. Lastly, inhibition of other upper glycolytic components, including PFKFB3/PFK with PFK158 or Hexokinase with Lonidamine, did not significantly affect RPE cell behavior. This study provides insights into the varied roles of upper glycolytic components in regulating the functionality of RPE cells. Specifically, it highlights the critical roles of Glut1 and Aldolase in preserving barrier integrity and promoting RPE cell adhesion and spreading. Such understanding will guide the development of safe interventions to treat RPE cell dysfunction in various retinal disorders.

Genipin ameliorates diabetic retinopathy via the HIF-1α and AGEs-RAGE pathways

BackgroundTraditional Chinese medicine (TCM) is useful in disease treatment and prevention. Genipin is an active TCM compound used to treat diabetic retinopathy (DR). In this study, a network pharmacology (NP)-based approach was employed to investigate the therapeutic mechanisms underlying genipin administration in DR. MethodsThe potential targets of DR were identified using the gene expression omnibus (GEO) database. TCM database screening and NP were used to predict the potential active targets and pathways of genipin in DR. Cell viability was tested in vitro to determine the effects of different doses of glucose and genipin on Human Retinal Microvascular Endothelial Cells (hRMECs). CCK-8, CCK-F, colony formation, CellTiter-Lum, Annexin V-FITC, wound healing, Transwell, tube-forming, reactive oxygen species (ROS), and other assay kits were used to detect the effects of genipin on hRMECs during high levels of glucose. In vivo, a streptozotocin (STZ)-mouse intraocular genipin injection (IOI.) model was used to explore the effects of genipin on diabetes-induced retinal dysfunction. Western blotting was performed to identify the cytokines involved in proliferation, apoptosis, angiogenesis, ROS, and inflammation. The protein expression of the AKT/ PI3K/ HIF-1α and AGEs/ RAGE pathways was also examined. ResultsApproximately 14 types of TCM, and nearly 300 active ingredients, including genipin, were identified. The NP approach successfully identified the HIF-1α and AGEs-RAGE pathways, with the EGR1 and UCP2 genes, as key targets of genipin in DR. In the in vitro and in vivo models, we discovered that high glucose increased cell proliferation, apoptosis, angiogenesis, ROS, and inflammation. However, genipin application regulated cell proliferation and apoptosis, inhibited angiogenesis, and reduced ROS and inflammation in the HRMECs exposed to high glucose. Furthermore, the retinal thickness in the genipin-treated group was lower than that in the untreated group. AKT/ PI3K/ HIF-1α and AGEs/ RAGE signaling was increased by high glucose levels; however, genipin treatment decreased AKT/ PI3K and AGEs/ RAGE pathway expressions. Genipin also increased HIF-1α phosphorylation, oxidative phosphorylation of ATP synthesis, lipid peroxidation, and the upregulation of oxidoreductase. Genipin was found to protect HG-induced hRMECs and the retina of STZ-mice, based on; 1 the inhibition of UCP2 and Glut1 decreased intracellular glucose, and glycosylation; 2 the increased presence of HIF-1α, which increased oxidative phosphorylation and decreased substrate phosphorylation; 3 the increase in oxidative phosphorylation from ATP synthesis increased lipid peroxidation and oxidoreductase activity, and; 4 the parallel effect of phosphorylation and glycosylation on vascular endothelial growth factor (VEGF), MMP9, and Scg3. ConclusionBased on NP, we demonstrated the potential targets and pathways of genipin in the treatment of DR and confirmed its effective molecular mechanism in vitro and in vivo. Genipin protects cells and tissues from high glucose levels by regulating phosphorylation and glycosylation. The activation of the HIF-1α pathway can also be used to treat DR. Our study provides new insights into the key genes and pathways associated with the prognosis and pathogenesis of DR.

Metabolic classification suggests the GLUT1/ALDOB/G6PD axis as a therapeutic target in chemotherapy-resistant pancreatic cancer

Metabolic reprogramming is known as an emerging mechanism of chemotherapy resistance, but the metabolic signatures of pancreatic ductal adenocarcinomas (PDACs) remain unclear. Here, we characterize the metabolomic profile of PDAC organoids and classify them into glucomet-PDAC (high glucose metabolism levels) and lipomet-PDAC (high lipid metabolism levels). Glucomet-PDACs are more resistant to chemotherapy than lipomet-PDACs, and patients with glucomet-PDAC have a worse prognosis. Integrated analyses reveal that the GLUT1/aldolase B (ALDOB)/glucose-6-phosphate dehydrogenase (G6PD) axis induces chemotherapy resistance by remodeling glucose metabolism in glucomet-PDAC. Increased glycolytic flux, G6PD activity, and pyrimidine biosynthesis are identified in glucomet-PDAC with high GLUT1 and low ALDOB expression, and these phenotypes could be reversed by inhibiting GLUT1 expression or by increasing ALDOB expression. Pharmacological inhibition of GLUT1 or G6PD enhances the chemotherapy response of glucomet-PDAC. Our findings uncover potential metabolic heterogeneity related to differences in chemotherapy sensitivity in PDAC and develop a promising pharmacological strategy for patients with chemotherapy-resistant glucomet-PDAC through the combination of chemotherapy and GLUT1/ALDOB/G6PD axis inhibitors.

Metformin exerts antileukemic effects by modulating lactate metabolism and overcomes imatinib resistance in chronic myelogenous leukemia.

Imatinib is the frontline treatment option in treating chronic myelogenous leukemia (CML). Hitherto, some patients relapse following treatment. Biochemical analysis of a panel of clonally derived imatinib-resistant cells revealed enhanced glucose uptake and ATP production, suggesting increased rates of glycolysis. Interestingly, increased lactate export was also observed in imatinib-resistant cell lines. Here, we show that metformin inhibits the growth of imatinib-resistant cell lines as well as peripheral blood mononuclear cells isolated from patients who relapsed following imatinib treatment. Metformin exerted these antiproliferative effects by inhibiting MCT1 and MCT4, leading to the inhibition of lactate export. Furthermore, glucose uptake and ATP production were also inhibited following metformin treatment due to the inhibition of GLUT1 and HK-II in an AMPK-dependent manner. Our results also confirmed that metformin-mediated inhibition of lactate export and glucose uptake occurs through the regulation of mTORC1 and HIF-1α. These results delineate the molecular mechanisms underlying metabolic reprogramming leading to secondary imatinib resistance and the potential of metformin as a therapeutic option in CML.

F.nucleatum facilitates oral squamous cell carcinoma progression via GLUT1-driven lactate production.

Tumor-resident microbiota has been documented for various cancer types. Oral squamous cell carcinoma (OSCC) is also enriched with microbiota, while the significance of microbiota in shaping the OSCC microenvironment remains elusive. We used bioinformatics and clinical sample analysis to explore relationship between F.nucleatum and OSCC progression. Xenograft tumor model, metabolic screening and RNA sequencing were performed to elucidate mechanisms of pro-tumor role of F.nucleatum. We show that a major protumorigenic bacterium, F.nucleatum, accumulates in invasive margins of OSCC tissues and drives tumor-associated macrophages (TAMs) formation. The mechanistic dissection shows that OSCC-resident F.nucleatum triggers the GalNAc-Autophagy-TBC1D5 signaling, leading to GLUT1 aggregation in the plasma membrane and the deposition of extracellular lactate. Simultaneous functional inhibition of GalNAc and GLUT1 efficiently reduces TAMs formation and restrains OSCC progression. These findings suggest that tumor-resident microbiota affects the immunomodulatory and protumorigenic microenvironment via modulating glycolysis and extracellular lactate deposition. The targeted intervention of this process could provide a distinct clinical strategy for patients with advanced OSCC. This work was supported by the National Natural Science Foundation of China for Key Program Projects (82030070, to LC) and Distinguished Young Scholars (31725011, to LC), as well as Innovation Team Project of Hubei Province (2020CFA014, to LC).

Warburg Effect as a Novel Mechanism for Homocysteine-Induced Features of Age-Related Macular Degeneration.

Age-related macular degeneration (AMD) is a major cause of blindness. Recent studies have reported impaired glycolysis in AMD patients with a high lactate/pyruvate ratio. Elevated homocysteine (Hcy) (Hyperhomocysteinemia, HHcy) was observed in several clinical studies, reporting an association between HHcy and AMD. We established the effect of HHcy on barrier function, retinal pigment epithelium (RPE) structure, and induced choroidal neovascularization (CNV) in mice. We hypothesize that HHcy contributes to AMD by inducing a metabolic switch in the mitochondria, in which cells predominantly produce energy by the high rate of glycolysis, or "Warburg", effect. Increased glycolysis results in an increased production of lactate, cellular acidity, activation of angiogenesis, RPE barrier dysfunction, and CNV. Evaluation of cellular energy production under HHcy was assessed by seahorse analysis, immunofluorescence, and western blot experiments. The seahorse analysis evaluated the extracellular acidification rate (ECAR) as indicative of glycolysis. HHcy showed a significant increase in ECAR both in vivo using (Cystathionine β-synthase) cbs+/- and cbs-/- mice retinas and in vitro (Hcy-treated ARPE-19) compared to wild-type mice and RPE cells. Moreover, HHcy up-regulated glycolytic enzyme (Glucose transporter-1 (GlUT-1), lactate dehydrogenase (LDH), and hexokinase 1 (HK1)) in Hcy-treated ARPE-19 and primary RPE cells isolated from cbs+/+, cbs+/-, and cbs-/- mice retinas. Inhibition of GLUT-1 or blocking of N-methyl-D-aspartate receptors (NMDAR) reduced glycolysis in Hcy-treated RPE and improved albumin leakage and CNV induction in Hcy-injected mice eyes. The current study suggests that HHcy causes a metabolic switch in the RPE cells from mitochondrial respiration to glycolysis during AMD and confirms the involvement of NMDAR in this process. Therefore, targeting Glycolysis or NMDAR could be a novel therapeutic target for AMD.

Inhibition of Cardiac Glucose Transport Corrects Diabetic Cardiomyopathy Even Without Alleviation of Hyperglycemia

Healthy hearts rely more on fatty acid (FA) rather than glucose utilization. It remains unclear whether diabetic cardiomyopathy (DbCM) is accounted for by glucotoxicity or lipotoxicity. Previously, we discovered that either insulin deficiency or insulin resistance causes FOXO1‐KLF5 activation in human and murine hearts, which drives cardiac lipotoxicity and oxidative stress. Now, we investigate how glucose activates cardiac KLF5 and causes DbCM. We hypothesized that higher cardiac glucose content in diabetes potentiates FOXO1‐KLF5 activation and causes glucolipotoxicity.We mimicked Type‐1 diabetes (T1D) in C57BL/6 mice via 5 daily intraperitoneal injections of streptozotocin (STZ). In contrast to late T1D (12 Wks post‐STZ), cardiac KLF5 expression levels are not increased in the early T1D (4 Wks post‐STZ). However, mice developed cardiac dysfunction in early T1D. Seahorse analysis in adult cardiomyocytes isolated from mice with early T1D showed suppression of FA dependence in the expense of higher glucose dependence compared to non‐diabetic mice. To confirm whether hyperglycemia or higher cardiac glucose content accounts for cardiac dysfunction in early T1D, we applied anti‐hyperglycemia treatment (Dapagliflozin, DAPA, SGLT2 inhibitor) or GLUT1 inhibition (STF‐31). Either of the two treatments restored cardiac FA dependence and prevented both glucose preference and cardiac dysfunction in early T1D. GC‐MS analysis in hearts of mice with late T1D, which have shifted back to increased FA dependence, showed increased cardiac glucose content ‐presumably unused glucose‐ and subsequent increase of cardiac KLF5, as previously shown, which exacerbates cardiac dysfunction. New data revealed that GLUT1 levels were increased in late T1D compared to early T1D, as well as that this is driven by KLF5 activation. The lack of activation of KLF5 expression in early T1D, which reverses in late T1D, is mirrored by FOXO1 transcriptional activity as shown by expression of FOXO1 targets and lower FOXO1 acetylation in late T1D, which is controlled by Sirtuin‐1. Analysis of hearts from mice with late T1D and a human cardiomyocyte cell line (AC16) that was treated with high glucose indicated higher Sirtuin‐1 expression and Sirtuin‐1 binding on FOXO1. Accordingly, prevention of glucose transport to hearts of diabetic mice via treatment with either DAPA or STF‐31 for 12 Wks lowered cardiac expression of KLF5 and its targets and improved cardiac function.Conclusively, DbCM begins in early T1D with lower mitochondrial FA utilization that is compensated by higher glucose utilization and is exacerbated in late T1D with activation of SIRT1‐FOXO1‐KLF5 axis that causes combined lipotoxicity and glucotoxicity. Inhibition of GLUT1 alleviates DbCM via prevention of both early glucose dependence and late KLF5 activation.

FSH mediates estradiol synthesis in hypoxic granulosa cells by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 signaling pathway

Owing to the avascular environment within ovarian follicles, granulosa cells (GCs) are believed to live in a hypoxic niche. Follicle-stimulating hormone (FSH)-mediated steroidogenesis is crucial for normal growth and maturation of ovarian follicles, but it remains unclear how FSH stimulates estradiol (E2) synthesis under hypoxic conditions. Here, we aimed to explore whether FSH affects the ATP production required for estrogen synthesis from the perspective of glucose metabolism. It was observed that the levels of both E2 and HIF-1α were markedly increased in a dose-dependent manner in mouse ovarian GCs after the injection of FSH in vivo, indicating that hypoxia/HIF-1α may be relevant to FSH-induced E2 synthesis. By treating hypoxic GCs with FSH in vitro, we further revealed that the activation of the AMP-activated protein kinase (AMPK)–GLUT1 pathway, which in turn stimulates ATP generation, may be essential for FSH-mediated E2 production during hypoxia. In contrast, inhibition of AMPK or GLUT1 with siRNAs/antagonist both repressed glycolysis, ATP production, and E2 synthesis despite FSH treatment. Moreover, blocking HIF-1α activity using siRNAs/PX-478 suppressed AMPK activation, GLUT1 expression, and E2 levels in FSH-treated GCs. Finally, the in vitro findings were verified in vivo, which showed markedly increased AMPK activity, GLUT1 expression, glycolytic flux, ATP levels, and E2 concentrations in ovarian GCs following FSH injection. Taken together, these findings uncovered a novel mechanism for FSH-regulating E2 synthesis in hypoxic GCs by activating glycolytic metabolism through the HIF-1α–AMPK–GLUT1 pathway.

Quantification of 2-NBDG, a probe for glucose uptake, in GLUT1 overexpression in HEK293T cells by LC–MS/MS

The growth and proliferation of most cancer cells involve the excessive uptake of glucose mediated by glucose transporters. An effective strategy for cancer therapy has been to inhibit the GLUTs that are usually overexpressed in a variety of tumor cells. 2-NBDG is a GLUT1 substrate that can be used as a probe for GLUT1 inhibitors. An accurate and simple assay for 2-NBDG in a HEK293T cell model overexpressing GLUT1 was developed using liquid chromatography-tandem mass spectrometry. Chromatographic separation was achieved using a Xbridge® Amide column (3.5 μm, 2.1 mm × 150 mm, Waters) with acetonitrile-water containing 2 μM ammonium acetate (80:20, v/v) at a flow rate of 0.25 mL/min. Mass detection was conducted in the parallel reaction monitoring (PRM) mode. The calibration curve for 2-NBDG showed good linearity in the concentration range of 5–500 ng/mL with satisfactory precision, a relative standard deviation ranging from 2.92 to 9.59% and accuracy with a relative error ranging from −13.14 to 7.34%. This method was successfully applied to quantify the uptake of GLUT1-mediated 2-NBDG, and the results clearly indicated inhibition of GLUT1 by WZB117 and quercetin (two potent glucose transporter inhibitors) in the GLUT1-HEK293T cell model. This study provides a convenient and accurate method for high-throughput screening of selective and promising GLUT1 inhibitors.

A mixture of persistent organic pollutants detected in human follicular fluid increases progesterone secretion and mitochondrial activity in human granulosa HGrC1 cells

Disruption of granulosa cells (GCs), the main functional cells in the ovary, is associated with impaired female fertility. Epidemiological studies demonstrated that women have detectable levels of organic pollutants (e.g., perfluorooctanoate, perfluorooctane sulfonate, 2,2-dichlorodiphenyldichloroethylene, polychlorinated biphenyl 153, and hexachlorobenzene) in their follicular fluid (FF), and thus these compounds may directly affect the function of GCs in the ovary. Considering that humans are exposed to multiple pollutants simultaneously, we elucidated the effects of a mixture of endocrine-disrupting chemicals (EDCs) on human granulosa HGrC1 cells. The EDC mixture directly increased progesterone secretion by upregulating 3β-hydroxysteroid dehydrogenase (3βHSD) expression. Furthermore, the EDC mixture increased activity of mitochondria, which are the central sites for steroid hormone biosynthesis, and the ATP content. Unexpectedly, the EDC mixture reduced glucose transporter 4 (GLUT4) expression and perturbed glucose uptake; however, this did not affect the glycolytic rate. Moreover, inhibition of GLUT1 by STF-31 did not alter the effects of the EDC mixture on steroid secretion but decreased basal estradiol secretion. Taken together, our results demonstrate that the mixture of EDCs present in FF can alter the functions of human GCs by disrupting steroidogenesis and may thus adversely affect female reproductive health. This study highlights that the EDC mixture elicits its effects by targeting mitochondria and increases mitochondrial network formation, mitochondrial activity, and expression of 3βHSD, which is associated with the inner mitochondrial membrane.

AMPK phosphorylates PPARδ to mediate its stabilization, inhibit glucose and glutamine uptake and colon tumor growth

Peroxisome proliferator–activated receptor δ (PPARδ) is a nuclear receptor transcription factor that plays an important role in the regulation of metabolism, inflammation, and cancer. In addition, the nutrient-sensing kinase 5′AMP-activated protein kinase (AMPK) is a critical regulator of cellular energy in coordination with PPARδ. However, the molecular mechanism of the AMPK/PPARδ pathway on cancer progression is still unclear. Here, we found that activated AMPK induced PPARδ-S50 phosphorylation in cancer cells, whereas the PPARδ/S50A (nonphosphorylation mimic) mutant reversed this event. Further analysis showed that the PPARδ/S50E (phosphorylation mimic) but not the PPARδ/S50A mutant increased PPARδ protein stability, which led to reduced p62/SQSTM1-mediated degradation of misfolded PPARδ. Furthermore, PPARδ-S50 phosphorylation decreased PPARδ transcription activity and alleviated PPARδ-mediated uptake of glucose and glutamine in cancer cells. Soft agar and xenograft tumor model analysis showed that the PPARδ/S50E mutant but not the PPARδ/S50A mutant inhibited colon cancer cell proliferation and tumor growth, which was associated with inhibition of Glut1 and SLC1A5 transporter protein expression. These findings reveal a new mechanism of AMPK-induced PPARδ-S50 phosphorylation, accumulation of misfolded PPARδ protein, and inhibition of PPARδ transcription activity contributing to the suppression of colon tumor formation.

Cellular binding and uptake of fluorescent glucose analogs 2-NBDG and 6-NBDG occurs independent of membrane glucose transporters

The classical methods for determining glucose uptake rates in living cells involve the use of isotopically labeled 2-deoxy-d-glucose or 3-O-methyl-d-glucose, which enter cells via well-characterized membrane transporters of the SLC2A and SLC5A families, respectively. These classical methods, however, are increasingly being displaced by high-throughput assays that utilize fluorescent analogs of glucose. Among the most commonly used of these analogs are 2-NBDG and 6-NBDG, which contain a bulky 7-nitro-2,1,3-benzoxadiazol-4-yl-amino moiety in place of a hydroxy group on d-glucose. This fluorescent group significantly alters both the size and shape of these molecules compared to glucose, calling into question whether they actually enter cells by the same transport mechanisms. In this study, we took advantage of the well-defined glucose uptake mechanism of L929 murine fibroblasts, which rely exclusively on the Glut1/Slc2a1 membrane transporter. We demonstrate that neither pharmacologic inhibition of Glut1 nor genetic manipulation of its expression has a significant impact on the binding or uptake of 2-NBDG or 6-NBDG by L929 cells, though both approaches significantly impact [3H]-2-deoxyglucose uptake rates. Together these data indicate that 2-NBDG and 6-NBDG can bind and enter mammalian cells by transporter-independent mechanisms, which calls into question their utility as an accurate proxy for glucose transport.

GLUT1 inhibition blocks growth of RB1-positive triple negative breast cancer

Triple negative breast cancer (TNBC) is a deadly form of breast cancer due to the development of resistance to chemotherapy affecting over 30% of patients. New therapeutics and companion biomarkers are urgently needed. Recognizing the elevated expression of glucose transporter 1 (GLUT1, encoded by SLC2A1) and associated metabolic dependencies in TNBC, we investigated the vulnerability of TNBC cell lines and patient-derived samples to GLUT1 inhibition. We report that genetic or pharmacological inhibition of GLUT1 with BAY-876 impairs the growth of a subset of TNBC cells displaying high glycolytic and lower oxidative phosphorylation (OXPHOS) rates. Pathway enrichment analysis of gene expression data suggests that the functionality of the E2F pathway may reflect to some extent OXPHOS activity. Furthermore, the protein levels of retinoblastoma tumor suppressor (RB1) strongly correlate with the degree of sensitivity to GLUT1 inhibition in TNBC, where RB1-negative cells are insensitive to GLUT1 inhibition. Collectively, our results highlight a strong and targetable RB1-GLUT1 metabolic axis in TNBC and warrant clinical evaluation of GLUT1 inhibition in TNBC patients stratified according to RB1 protein expression levels.

Activated but Not Quiescent Hematopoietic Stem Cells Rely Readily on Glycolysis As Their Main Source of Energy

Elucidating mechanisms that regulate hematopoietic stem cells (HSCs) quiescence is critical for improving bone marrow transplantation. It is postulated that quiescent HSCs rely mostly on glycolysis rather than mitochondrial oxidative phosphorylation (OXPHOS) as their energy source. We have identified a population of HSCs with low mitochondrial activity (LSKCD150+CD48- within the 25% lowest mitochondrial membrane potential; MMP-low) that we show are highly enriched in quiescent HSCs (~95% ±2.65 in G0) as measured by pyronin Y/Hoechst staining in contrast to LSKCD150+CD48- within 25% highest MMP (MMP-high) that are in majority in G1/S/G2/M phase of cell cycle (55.4%±16; p<0.05; n=3). MMP-low HSCs exhibit greater in vivo competitive repopulation ability (3.7 fold; p=0.021; n=10 mice) at 16 weeks as compared to MMP-high fractions, show higher self renewal ability and are enriched in label-retaining H2B-GFP+ cells (~3 fold; p<0.01; n=3). Conversely, label-retaining GFP+HSCs maintain lower MMP than non-label-retaining cells. Altogether these results reinforce the notion that MMP-low HSCs are quiescent whereas MMP-high HSCs are primed/activated. Using single cell RNA-sequencing (scRNA-seq) analysis to interrogate the transcriptome by a Fluidigm C1 platform within MMP-high versus MMP-low HSCs we found major metabolic pathways including OXPHOS, exhibit significantly greater expression within the MMP-high than in the MMP-low HSC fraction. Seahorse analysis confirmed that MMP-high but not -low hematopoietic stem and progenitor cells (HSPCs) use OXPHOS as their source of energy (3.9 fold; n=16 mice). Unexpectedly, glycolytic gene expression was also enriched in the primed MMP-high HSCs and relatively low in quiescent MMP-low HSCs. qRT-PCR analysis further confirmed that the expression of glycolytic enzymes and other genes including glucose transporter 1 (Glut1, slc2a1) that is the main glucose transporter on HSCs is greater in MMP-high relative to -low HSCs. These unforeseen findings raised the potential that despite the current consensus in HSC biology, glycolysis may more readily support activated rather than quiescent HSCs. We thus measured the glucose uptake in MMP-low vs -high HSCs under metabolic (pyruvate, glucose and glutamine)-restricted conditions. Using 2NBDG, a fluorescently tagged glucose analog, we found that MMP-high HSCs uptake 3.3-fold more glucose and contained three times more 2NBDG+ cells as compared to MMP-low HSCs (p<0.001 for each; n=3). Pharmacological inhibition of Glut1 reduced glucose uptake in MMP-high but not -low HSCs suggesting the specific sensitivity of MMP-high HSCs to the glucose inhibition. In addition, activation of tricarboxylic acid cycle TCA cycle with combined methyl-pyruvate and dimethyl-alpha-ketoglutarate led to a greater glucose uptake (~3.5 fold) in MMP-high as compared to MMP low HSCs (~2.2 fold; p<0.0001). To address the degree to which glycolysis is necessary, FACS-purified MMP-low and -high HSCs were incubated with 2-Deoxy D-Glucose (2DG) - a glucose analog - that inhibits glycolysis via its action on hexokinase. While glucose deprivation with 50 mM dose of 2DG within 12 hours did not have much effect on MMP-low HSCs, over 60% of MMP-high HSCs died (p<0.001; n=3), suggesting that MMP-high but not -low HSCs rely readily on glycolysis for their survival. Also, the inhibition of mitochondrial transport of pyruvate that is the end product of glycolysis (α-cyano-4-hydroxycinnamate (CHC)), decreased survival in MMP-high HSCs by 80% with negligible effect on MMP-low HSCs (p<0.01; n=3). Finally 50 FACS-purified HSCs (LSKCD150+CD48-MMP-low or -high) were transplanted in lethally irradiated mice along with 200,000 unfractionated bone marrow cells in a competitive repopulation assay and mice were subsequently treated with 2DG or control vehicle every other day for two weeks. Two months later, 2DG treatment led to significantly enhanced reconstitution levels in MMP-high HSCs with no or little effect on MMP low reconstitution levels (ongoing). These combined results suggest that primed MMP-high rather than quiescent MMP-low HSCs rely on glycolysis as their main source of energy. These findings are consistent with the concept that glycolysis is key in sustaining rapidly dividing cells such as embryonic stem cells and cancer cells. Disclosures Ghaffari: Rubius Therapeutics: Consultancy.

Dual Inhibition of GLUT1 and the ATR/CHK1 Kinase Axis Displays Synergistic Cytotoxicity in KRAS-Mutant Cancer Cells.

The advent of molecularly targeted therapeutic agents has opened a new era in cancer therapy. However, many tumors rely on nondruggable cancer-driving lesions. In addition, long-lasting clinical benefits from single-agent therapies rarely occur, as most of the tumors acquire resistance over time. The identification of targeted combination regimens interfering with signaling through oncogenically rewired pathways provides a promising approach to enhance efficacy of single-agent-targeted treatments. Moreover, combination drug therapies might overcome the emergence of drug resistance. Here, we performed a focused flow cytometry-based drug synergy screen and identified a novel synergistic interaction between GLUT1-mediated glucose transport and the cell-cycle checkpoint kinases ATR and CHK1. Combined inhibition of CHK1/GLUT1 or ATR/GLUT1 robustly induced apoptosis, particularly in RAS-mutant cancer cells. Mechanistically, combined inhibition of ATR/CHK1 and GLUT1 arrested sensitive cells in S-phase and led to the accumulation of genotoxic damage, particularly in S-phase. In vivo, simultaneous inhibition of ATR and GLUT1 significantly reduced tumor volume gain in an autochthonous mouse model of KrasG12D -driven soft tissue sarcoma. Taken together, these findings pave the way for combined inhibition of GLUT1 and ATR/CHK1 as a therapeutic approach for KRAS-driven cancers. SIGNIFICANCE: Dual targeting of the DNA damage response and glucose transport synergistically induces apoptosis in KRAS-mutant cancer, suggesting this combination treatment for clinical validation in KRAS-stratified tumor patients.

Elevated UCP1 levels are sufficient to improve glucose uptake in human white adipocytes

Brown adipose tissue (BAT) has been considered beneficial for metabolic health by participating in the regulation of glucose homoeostasis. The browning factors that improve glucose uptake beyond normal levels are still unknown but glucose uptake is not affected in UCP1 knockout mice. Here, we demonstrate in human white adipocytes that basal/resting glucose uptake is improved by solely elevating UCP1 protein levels. Generating human white Simpson-Golabi-Behmel syndrome (SGBS) adipocytes with a stable knockout and overexpression of UCP1, we discovered that UCP1 overexpressing adipocytes significantly improve glucose uptake by 40%. Mechanistically, this is caused by higher glycolytic flux, seen as increased oxygen consumption, extracellular acidification and lactate secretion rates. The improvements in glucose handling are comparable to white-to-brown transitions, as judged by, for the first time, directly comparing in vitro differentiated mouse brown vs white adipocytes. Although no adipogenic, metabolic and mitochondrial gene expressions were significantly altered in SGBS cells, pharmacological inhibition of GLUT1 completely abrogated differences between UCP1+ and control cells, thereby uncovering GLUT1-mediated uptake as permissive gatekeeper. Collectively, our data demonstrate that elevating UCP1 levels is sufficient to improve human white adipocytes as a glucose sink without adverse cellular effects, thus not requiring the adrenergic controlled, complex network of browning which usually hampers translational efforts.

Abstract TP515: A New Approach for Inhibiting Hyperglycolysis in Ischemic Stroke: Neuroprotective Effect of Pharmacological Hypothermia with Combined Phenothiazines [Chlorpromazine and Promethazine (C+P)] and Dihydrocapsaicin (DHC)

Introduction: Hyperglycolysis, the increase in glucose uptake and metabolism relative to the rate of oxygen utilization, is detrimental because of uncoupling of glycolysis, oxidative phosphorylation and thus lactic acidosis. Therapeutic hypothermia (TH) is one of the most effective neuroprotectants against ischemic injury. However, its clinical use in stroke is restricted due to slow onset, extensive medical and nursing efforts, and complications. Pharmacological hypothermia is an important alternative to induce TH. DHC is a powerful hypothermia inducing agent that targets brain thermoregulation. However, the complications associated with a high dose for achieving TH limited its use. Previous study has showed that C+P decreased brain activity and body temperature. We aimed to establish a therapeutic strategy of synergistic TH for faster cooling and stronger neuroprotection. Methods: Sprague-Dawley rats were subjected to 2 h middle cerebral artery occlusion (MCAO) and 6 or 24 h reperfusion, upon which rats intraperitoneally received very low dose of C+P (4 mg/kg), DHC (0.5 mg/kg) or their combination, or inhibitors of glucose transporter 1 and 3 (GLU1/3) (phloretin/cyclin B). Infarct volumes and neurological deficits were examined. Oxidative injury was determined by levels of ATP, lactate, and reactive oxygen species (ROS). Hyperglycolysis was determined by brain glucose, as well as the protein levels of GLUT1, GLUT3, PFK and LDH. Results: Body temperature reached the lowest target at 36.5°C by DHC, 35.2°C by C+P, 34°C by combination. Following 6 and 24 h of reperfusion, combination therapy exhibited greater reduction of infarct volume, neurological deficit, brain glucose, lactate and ROS levels, but increased ATP compared to monotherapy. Meanwhile, both DHC and C+P monotherapies reduced GLUT1, GLUT3, PFK and LDH expression, and the reduced expression of these proteins was enhanced by combination therapy. Inhibition of GLUT1 and GLUT3 showed similar trend as the combination therapy group. Conclusion: The combination of C+P and DHC enhances the efficiency of TH and efficacy of TH-induced neuroprotection in ischemic stroke. This may be mediated by inhibiting glucose metabolism and associated hyperglycolysis.

Involvement of GLUT1-mediated glucose transport and metabolism in gefitinib resistance of non-small-cell lung cancer cells.

Use of epidermal growth factor receptor (EGFR) inhibitors represented by gefitinib and erlotinib has become the standard of treatment for non-small-cell lung cancers (NSCLCs) with activating EGFR mutations. However, the majority of NSCLCs, which overexpress EGFR without such mutations, are resistant to EGFR inhibitors, and the mechanism(s) behind such primary resistance of NSCLCs without activating EGFR mutations to EGFR inhibitors still remains poorly understood. Here in this study, we show that glucose metabolism mediated by GLUT1, a facilitative glucose transporter, is involved in gefitinib resistance of NSCLC cells. We found that GLUT1 expression and glucose uptake were increased in resistant NSCLC cells after gefitinib treatment and that genetic as well as pharmacological inhibition of GLUT1 sensitized not only NSCLC cells with primary resistance but also those with acquired resistance to gefitinib. In vivo, the combination of systemic gefitinib and a GLUT1 inhibitor, both of which failed to inhibit tumor growth when administered alone, significantly inhibited the growth of xenograft tumors formed by the implantation of NSCLC cells with wild-type EGFR (wt-EGFR). Since our data indicated that GLUT1 was similarly involved in erlotinib resistance, our findings suggest that the activity of GLUT1-mediated glucose metabolism could be a critical determinant for the sensitivity of NSCLC cells to EGFR inhibitors and that concurrent GLUT1 inhibition may therefore be a mechanism-based approach to treating NSCLCs resistant to EGFR inhibitors, including those with wt-EGFR.

Effect of metformin on estrogen and progesterone receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cells

This work aimed to investigate the effect of metformin on cellular glucose uptake and metabolism by breast cancer cells, as a mechanism contributing to its anticancer properties. Estrogen and progesterone receptor-positive (MCF-7) and triple-negative (MDA-MB-231) breast cancer cell lines were used as in vitro models of breast cancer.Short-term (26 min) exposure of MCF-7 and MDA-MB-231 cells to metformin inhibited uptake of 3H-deoxy-D-glucose (3H-DG). In contrast, long-term (24 h) exposure to metformin (5 μM–1 mM) concentration-dependently increased 3H-DG uptake in both cell lines. This effect was associated with an increase in lactate production but was not associated with changes in GLUT1 mRNA expression. Long-term exposure of MCF-7 and MDA-MB-231 cells to metformin (5 μM–1 mM) concentration-dependently reduced cell viability and culture mass and slightly increased cell proliferation rates. Combination of metformin (1 mM) with the facilitative glucose transporter (GLUT) inhibitor kaempferol (30 μM) did not change the effect of metformin on culture growth.In conclusion, short-term exposure to metformin reduces cellular glucose uptake, probably by direct inhibition of GLUT1. However, after long-term exposure to metformin, cellular uptake of glucose is significantly increased, not associated to changes in GLUT1 transcription rates. We suggest that, in the long-term, metformin induces a compensatory increase in glucose uptake in response to cellular energy depletion resulting from its inhibitory effect on mitochondrial oxidative phosphorylation machinery. Metformin-induced dependence of breast cancer cells on glycolytic pathway, associated with an anticarcinogenic effect of the drug, provides a biochemical basis for the design of new therapeutic strategies.

Abstract 4731: Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer

Abstract Oncogenic KRAS signaling is the main driving force behind pancreatic ductal adenocarcinoma (PDAC); however, targeting this pathway has proven to be difficult. Conversely, the PI3K/Akt pathway represents an exciting new target, because it has been associated with poor prognosis and chemoresistance, and several inhibitors are under development. In particular, perifosine prevents Akt translocation to the cell membrane, while MK-2206 is an Akt allosteric inhibitor and BEZ-235 is a dual PI3K/mTOR inhibitor. Therefore, we investigated the prognostic role of phospho (p)-Akt in PDAC tissues, as well as the molecular mechanisms underlying the interaction of Akt inhibitors with gemcitabine, using PDAC cells, primary cultures and spheroids. Immunohistochemistry of tissue microarrays with specimens from radically-resected patients (n=100) revealed a correlation between high p-Akt1 expression and worse outcome. Patients with low p-Akt expression (as detected by digital scoring) had a median overall survival (OS) of 16.2 months (95% CI, 14.8-20.1), while patients with high expression had a median OS of 12.0 months (95% CI, 9.0-14.9, P=0.03). Parallel immunocytochemistry studies revealed high expression levels in LPC028 primary cells, while LPC006 were characterized by low p-Akt1. Akt inhibitors reduced cancer cell growth in monolayers and spheroids, and synergistically enhanced the antiproliferative activity of gemcitabine in LPC028 (e.g., combination index CI of 0.2, in the gemcitabine-perifosine combination for 72h, at fixed IC50 ratio), while this combination was antagonistic in LPC006 cells. The synergistic effect was paralleled by a 5-fold reduction in the expression of the main gemcitabine target ribonucleotide reductase. Inhibition of Akt decreased cell migration and invasion, which was additionally reduced by the combination with gemcitabine. However, the combination of Akt inhibitors with gemcitabine increased apoptosis, associated with induction of caspase-3/6/8/9, PARP and BAD, and inhibition of Bcl-2 and NF-kB in LPC028, but not in LPC006 cells. The Akt signaling is involved in the expression/localization of the key glucose transporter Glut1, and increased glucose metabolism was associated to resistance to axitinib (Hudson et al, Cell Death Dis 2014). Remarkably, the resistant LPC006 cells were characterized by overexpression of Glut1, which was not reduced after exposure to Akt inhibitors. However, the novel Glut1 inhibitor PGL13 enhanced perifosine and perifosine/gemcitabine-induced cell death. In conclusion, our findings support the analysis of phospho-Akt1 expression as both a prognostic and a predictive biomarker, for the rational development of new combination therapies targeting the Akt pathway in PDAC. Finally, inhibition of Glut1 might overcome resistance to these therapies and warrants further studies. Citation Format: Daniela Massihnia, Amir Avan, Niccola Funel, Carlotta Granchi, Filippo Minutolo, Leticia Leon, Godefridus Peters, Elisa Giovannetti. Phospho-akt: a potential resistance marker to chemotherapy and a therapy-target to restore sensitivity in pancreatic cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 4731. doi:10.1158/1538-7445.AM2017-4731