GLUT1 Inhibitor Research Articles

Highlights from Recent Cancer Literature

Highlights from Recent Cancer Literature

Influences of galactose ligand on the uptake of TADF liposomes by HepG2 cells.

Glucose is the main energy substance to drive the physiological events of the cell.. Malignant cells exhibit a much higher rate of glycolysis than healthy cells to relieve the increased needs of energy. The higher metabolic rate induces the over-expression of the Glucose Transporter (GLUT) to transport more glucose into malignant cells. Our research regarded overexpressive GLUT as a target of nanoparticles. Substrate of GLUT galactose conjugated Polyethylene glycol-Distearyl phosphatidyl ethanolamine (PEG-DSPE) as a kind of ligand was selected to modified liposome. Thermally activated delayed fluorescence (TADF) was encapsulated as fluorescent probe to evaluate its abilities of targeting malignant cells, and the results of confocal microscopy and flow cytometry demonstrated that Galactose-PEG-DSPE modified liposome had the stronger efficiency of cellular uptake by HepG2 cells compared with Blank-PEG-DSPE modified liposome. The effect of GLUT1 inhibitor on cellular uptake of Galactose-PEG-DSPE modified liposomes showed that the mechanism might be relative to Warburg effect causing GLUT overexpression.

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.

Effect of Addition of WZB117 as an Inhibitor of Glucose Transporter 1 for Venous Blood Glucose Determination.

Sodium fluoride (NaF) has been applied to inhibit glycolysis in venous specimens for decades. However, it has had little effect on the rate of glycolysis in the first 1 to 2 hours, resulting in a decrease of glucose, so a more efficient method is needed. Recently, we discovered that WZB117, a specific Glut1 inhibitor, restricts glycolysis by inhibiting the passive sugar transport of human red blood cells and cancer cells. The purpose of this study was to evaluate the results of intravenous blood glucose determination after the addition of WZB117. Venous specimens from 40 pairs of healthy volunteers were collected for several days and placed in tubes containing NaF plus EDTA-disodium (Na2) without WZB117 (the A group); citric acid, trisodium citrate, and EDTA-Na2 without WZB117 (B group); and NaF plus EDTA-Na2 with WZB117 (C group). The glucose concentration was measured after venipuncture and compared with test tubes treated for 1 hour, 2 hours, and 3 hours before centrifugation. Glucose level was determined by the hexokinase method. The paired t-test was used to examine differences in glucose values at baseline and at different time points. The number of misdiagnoses and the misdiagnosis rate were calculated at 2 diagnostic stages: high risk of diabetes (glucose level of 6.1 mmol/L) and diagnosis of diabetes (glucose level of 7.0 mmol/L). Glucose levels decreased by 1.0% at 1 hour and by 2.1% at 3 hours in the C group tubes and simultaneously decreased by 1.7% at 1 hour and by 2.5% at 3 hours in the B group tubes. In contrast, glucose levels decreased by 4.1% at 1 hour and by 6.3% at 3 hours in the A group tubes. There was a statistically significant difference in glucose levels measured in the A group tubes and B group tubes at 1 hour, 2 hours, and 3 hours. The misdiagnosis rate of clinical diagnosis in diabetes was highest in the A group tubes (7.0‰ at 1 hour, 0.1‰ at 3 hours at 7.0 mmol/L point; 14.6‰ at 1 hour, 0.4‰ at 3 hours at 6.1 mmol/L point) and lowest in the C group tubes (2.95‰ at 1 hour, 0‰ at 3 hours at 7.0 mmol/L point; 4.8‰ at 1 hour, 0.1‰ at 3 hours at 6.1 mmol/L point). The tube addition of WZB117 is more suitable for minimizing glycolysis and has no effect on glucose levels even if specimens are left uncentrifuged for up to 3 hours.

Structure guided design and synthesis of furyl thiazolidinedione derivatives as inhibitors of GLUT 1 and GLUT 4, and evaluation of their anti-leukemic potential

Cancer cells increase their glucose uptake and glycolytic activity to meet the high energy requirements of proliferation. Glucose transporters (GLUTs), which facilitate the transport of glucose and related hexoses across the cell membrane, play a vital role in tumor cell survival and are overexpressed in various cancers. GLUT1, the most overexpressed GLUT in many cancers, is emerging as a promising anti-cancer target. To develop GLUT1 inhibitors, we rationally designed, synthesized, structurally characterized, and biologically evaluated in-vitro and in-vivo a novel series of furyl-2-methylene thiazolidinediones (TZDs). Among 25 TZDs tested, F18 and F19 inhibited GLUT1 most potently (IC50 11.4 and 14.7 μM, respectively). F18 was equally selective for GLUT4 (IC50 6.8 μM), while F19 was specific for GLUT1 (IC50 152 μM in GLUT4). In-silico ligand docking studies showed that F18 interacted with conserved residues in GLUT1 and GLUT4, while F19 had slightly different interactions with the transporters. In in-vitro antiproliferative screening of leukemic/lymphoid cells, F18 was most lethal to CEM cells (CC50 of 1.7 μM). Flow cytometry analysis indicated that F18 arrested cell cycle growth in the subG0-G1 phase and lead to cell death due to necrosis and apoptosis. Western blot analysis exhibited alterations in cell signaling proteins, consistent with cell growth arrest and death. In-vivo xenograft study in a CEM model showed that F18 impaired tumor growth significantly.

High fat diet altered cardiac metabolic gene profile in Psammomys obesus gerbils

BackgroundIn metabolic disorders, myocardial fatty infiltration is critically associated with lipotoxic cardiomyopathy.MethodsTwenty Psammomys obesus gerbils were randomly assigned to normal plant or high fat diet. Sixteen weeks later, myocardium was sampled for pathobiological evaluation.ResultsA sixteen-week high fat diet resulted in myocardial structure disorganization, with collagen deposits, lipid accumulation, cardiomyocyte apoptosis and inflammatory cell infiltration. Myocardial expressions of glucose transporter GLUT1 and pyruvate dehydrogenase (PDH) inhibitor, PDH kinase (PDK)4 increased, while insulin-regulated GLUT4 expression remained unchanged. Myocardial expressions of molecules regulating fatty acid transport, CD36 and fatty acid binding protein (FABP)3, were increased, while expression of rate-controlling fatty acid β-oxidation, carnitine palmitoyl transferase (CPT)1B decreased. Myocardial expression of AMP-activated protein kinase (AMPK), decreased, while expression of peroxisome proliferator activated receptors (PPAR)-α and -γ did not change.ConclusionIn high fat diet fed Psammomys obesus, an original experimental model of nutritionally induced metabolic syndrome mixing genetic predisposition and environment interactions, a short period of high fat feeding was sufficient to induce myocardial structural alterations, associated with altered myocardial metabolic gene expression in favor of lipid accumulation.

GLUT1 is an AR target contributing to tumor growth and glycolysis in castration-resistant and enzalutamide-resistant prostate cancers.

Castration-resistant prostate cancer (CRPC) displays a higher 18F-FDG PET SUVmax than hormone-sensitive prostate cancer, which suggests a greater need for glucose metabolism in CRPC. Targeting glucose metabolism in cancer cells remains attractive for cancer treatment. Glucose transporters (GLUTs) meditate the first and rate-limiting step of glucose metabolism. Here, we investigated the key mediator of glucose transporters and evaluated its therapeutic value in a preclinical model of CRPC. 18F-FDG PET showed a higher SUVmax in CRPC than in hormone-sensitive prostate cancer, and GLUT1 expression positively correlated with SUVmax and was associated with a worse CRPC outcome. GLUT1 inhibition significantly suppressed cell growth, glycolysis and tumor volume in a xenograft model both in CRPC and enzalutamide-resistant prostate cancer. Chromatin immunoprecipitation and dual luciferase reporter assay showed that androgen receptor (AR) directly bound to the GLUT1 gene promoter to promote GLUT1 transcription. Combining GLUT1 inhibition and enzalutamide remarkably suppressed proliferation and glycolysis and induced apoptosis in CRPC cells. Our results suggest that GLUT1 is an AR target and displays synergistic effects with enzalutamide. GLUT1 may act as a promising therapeutic target in CRPC and enzalutamide-resistant prostate cancer.

Fasentin diminishes endothelial cell proliferation, differentiation and invasion in a glucose metabolism-independent manner

The synthetic compound fasentin has been described as a modulator of GLUT-1 and GLUT-4 transporters, thus inhibiting glucose uptake in some cancer cells. Endothelial glucose metabolism has been recently connected to angiogenesis and it is now an emerging topic in scientific research. Indeed, certain compounds with a known effect on glucose metabolism have also been shown to inhibit angiogenesis. In this work we tested the capability of fasentin to modulate angiogenesis in vitro and in vivo. We show that fasentin inhibited tube formation in endothelial cells by a mechanism that involves a negative effect on endothelial cell proliferation and invasion, without affecting other steps related to the angiogenic process. However, fasentin barely decreased glucose uptake in human dermal microvascular endothelial cells and the GLUT-1 inhibitor STF-31 failed to inhibit tube formation in these cells. Therefore, this modulatory capacity on endothelial cells function exerted by fasentin is most likely independent of a modulation of glucose metabolism. Taken together, our results show a novel biological activity of fasentin, which could be evaluated for its utility in cancer and other angiogenesis-dependent diseases.

Ligand-based design of GLUT inhibitors as potential antitumor agents

Glucose transporters (GLUTs) regulate glucose uptake and are often overexpressed in several human tumors. To identify new chemotypes targeting GLUT1, we built a pharmacophore model and searched against a NCI compound database. Sixteen hit molecules with good docking scores were screened for GLUT1 inhibition and antiproliferative activities. From these, we identified that compounds 2, 5, 6 and 13 inhibited the cell viability in a dose-dependent manner and that the IC50s of 2 and 6 are<10 µM concentration in the HCT116 colon cancer cell line. Lead compound 13 (NSC295720) was a GLUT1 inhibitor. Docking studies show that GLUT1 residues Phe291, Phe379, Glu380, Trp388, and Trp412 were important for inhibitor binding.

Dysregulation of the miR-148a\u2013GLUT1 axis promotes the progression and chemoresistance of human intrahepatic cholangiocarcinoma

Intrahepatic cholangiocarcinoma (iCCA) is a highly fatal malignant cancer worldwide. Elucidating the underlying molecular mechanism of iCCA progression is critical for the identification of new therapeutic targets. The present study explored the role of the miR-148a–GLUT1 axis in the progression of iCCA. The expression of GLUT1 was detected by using immunohistochemistry, western blot assays, and real-time polymerase chain reaction. The effects of GLUT1 on cell proliferation, invasion, and chemoresistance were investigated both in vitro and in vivo. A luciferase reporter assay was used to explore the effect of miR-148a on GLUT1 expression. GLUT1 was overexpressed in iCCA tissues. GLUT1 overexpression was associated with shorter overall and disease-free survival. Knockdown of GLUT1 reduced, while overexpression of GLUT1 promoted, the proliferation, motility, and invasiveness of iCCA cells in vitro and in vivo. Silencing GLUT1 significantly sensitized iCCA cells to gemcitabine in vitro and in vivo. GLUT1 was directly regulated by miR-148a, whose downregulation was associated with the proliferation, migration, and invasion of iCCA cells. WZB117, a GLUT1 inhibitor, inhibited tumor growth in an iCCA patient-derived xenograft model. These results indicate that downregulation of miR-148a levels results in GLUT1 overexpression in iCCA, leading to iCCA progression and gemcitabine resistance.

The Combination of MK-2206 and WZB117 Exerts a Synergistic Cytotoxic Effect Against Breast Cancer Cells.

Breast cancer is the most commonly diagnosed cancer and the second leading cause of cancer death in women. Hormone receptor-positive breast cancer is usually subjected to hormone therapy, while triple-negative breast cancer is more formidable and poses a therapeutic challenge. Glucose transporters are potential targets for the development of anticancer drugs. In search of anticancer agents whose effect could be enhanced by a GLUT1 inhibitor WZB117, we found that MK-2206, a potent allosteric Akt inhibitor, when combined with WZB117, showed a synergistic effect on growth inhibition and apoptosis induction in breast cancer cells, including ER(+) MCF-7 cells and triple-negative MDA-MB-231 cells. The combination index values at 50% growth inhibition were 0.45 and 0.21, respectively. Mechanism studies revealed that MK-2206 and WZB117 exert a synergistic cytotoxic effect in both MCF-7 and MDA-MB-231 breast cancer cells by inhibiting Akt phosphorylation and inducing DNA damage. The combination may also compromise DNA damage repair and ultimately lead to apoptosis. Our findings suggest that the combination of Akt inhibitors and GLUT1 inhibitors could be a novel strategy to combat breast cancer.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays.

Glucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP-dependent kinase, inhibition of mTOR signaling, and induction of cell-cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Discovery of a Potent GLUT Inhibitor from a Library of Rapafucins by Using 3D Microarrays

AbstractGlucose transporters play an essential role in cancer cell proliferation and survival and have been pursued as promising cancer drug targets. Using microarrays of a library of new macrocycles known as rapafucins, which were inspired by the natural product rapamycin, we screened for new inhibitors of GLUT1. We identified multiple hits from the rapafucin 3D microarray and confirmed one hit as a bona fide GLUT1 ligand, which we named rapaglutin A (RgA). We demonstrate that RgA is a potent inhibitor of GLUT1 as well as GLUT3 and GLUT4, with an IC50 value of low nanomolar for GLUT1. RgA was found to inhibit glucose uptake, leading to a decrease in cellular ATP synthesis, activation of AMP‐dependent kinase, inhibition of mTOR signaling, and induction of cell‐cycle arrest and apoptosis in cancer cells. Moreover, RgA was capable of inhibiting tumor xenografts in vivo without obvious side effects. RgA could thus be a new chemical tool to study GLUT function and a promising lead for developing anticancer drugs.

Pharmacological Targeting of GLUT1 to Control Autoreactive T Cell Responses.

An increasing body of evidence indicates that bio-energetic metabolism of T cells can be manipulated to control T cell responses. This potentially finds a field of application in the control of the T cell responses in autoimmune diseases, including in type 1 diabetes (T1D). Of the possible metabolic targets, Glut1 gained considerable interest because of its pivotal role in glucose uptake to fuel glycolysis in activated T cells, and the recent development of a novel class of small molecules that act as selective inhibitor of Glut1. We believe we can foresee a possible application of pharmacological Glut1 blockade approach to control autoreactive T cells that destroy insulin producing beta cells. However, Glut1 is expressed in a broad range of cells in the body and off-target and side effect are possible complications. Moreover, the duration of the treatment and the age of patients are critical aspects that need to be addressed to reduce toxicity. In this paper, we will review recent literature to determine whether it is possible to design a pharmacological Glut1 blocking strategy and how to apply this to autoimmunity in T1D.

The Progress and Development of GLUT1 Inhibitors Targeting Cancer Energy Metabolism

A major difference between glucose metabolism in cancer cells and normal cells is that glucose in cancer cells is preferably converted to lactate in aerobic conditions rather than oxidized in mitochondria. This process is called aerobic glycolysis, known as the 'Warburg effect'. In this review, we focus on the energy-metabolism characteristics between tumor and normal cells, analyzing the regulation mechanism of energy metabolism based on glycolysis, and summarizing two targets on the upstream proteins of glycolysis, including glucose transporter (GLUT) and hexokinase. In addition, we proposed the risks and limitations of GLUT1-based drug research and summarized the current research progress of representative drugs, including natural and synthetic GLUT1 inhibitors. This will provide guidance for designing and synthesizing small molecule drugs targeting GLUT1 in glycolysis.

Erianin alleviates diabetic retinopathy by reducing retinal inflammation initiated by microglial cells via inhibiting hyperglycemia-mediated ERK1/2-NF-κB signaling pathway.

Blood-retinal barrier (BRB) breakdown is a typical event in the early stage of diabetic retinopathy (DR). This study aims to elucidate the protection of erianin, a natural compound isolated from Dendrobium chrysotoxum Lindl, against DR development. Erianin alleviated BRB breakdown and rescued the reduced claudin1 and occludin expression in retinas from streptozotocin-induced diabetic mice. Erianin reduced microglial activation, ERK1/2 phosphorylation, NF-κB transcriptional activation, and the elevated TNF-α expression both in vitro and in vivo. ERK1/2 inhibitor U0126 abrogated NF-κB activation in d-glucose-treated BV2 cells. Erianin reduced cellular glucose uptake, and molecular docking analysis indicated the potential interaction of erianin with glucose transporter (GLUT)1. GLUT1 inhibitor (STF31) reduced the activation of the ERK1/2-NF-κB signaling pathway. Coculture with d-glucose-stimulated microglial BV2 cells and with TNF-α stimulation both induced inner BRB and outer BRB damage in human retinal endothelial cells and APRE19 cells, but erianin improved all these damages. In summary, erianin attenuated BRB breakdown during DR development by inhibiting microglia-triggered retinal inflammation via reducing cellular glucose uptake and abrogating the subsequent activation of the downstream ERK1/2-NF-κB pathway. Moreover, erianin also alleviated BRB damage induced by TNF-α released from the activated microglia.-Zhang, T., Ouyang, H., Mei, X., Lu, B., Yu, Z., Chen, K., Wang, Z., Ji, L. Erianin alleviates diabetic retinopathy by reducing retinal inflammation initiated by microglial cells via inhibiting hyperglycemia-mediated ERK1/2-NF-κB signaling pathway.

Sugar Modification Enhances Cytotoxic Activity of PAMAM-Doxorubicin Conjugate in Glucose-Deprived MCF-7 Cells \u2013 Possible Role of GLUT1 Transporter

PurposeIn order to overcome the obstacles and side effects of classical chemotherapy, numerous studies have been performed to develop the treatment based on targeted transport of active compounds directly to the site of action. Since tumor cells are featured with intensified glucose metabolism, we set out to develop innovative, glucose-modified PAMAM dendrimer for the delivery of doxorubicin to breast cancer cells.MethodsPAMAM-dox-glc conjugate was synthesized and characterized by 1H NMR, FT-IR, size and zeta potential measurements. The drug release rate from conjugate was evaluated by dialysis under different pH conditions. The expression level of GLUT family receptors in cells cultured in full and glucose-deprived medium was evaluated by quantitative real-time RT-PCR and flow cytometry. The cytotoxicity of conjugate in presence or absence of GLUT1 inhibitors was determined by MTT assay.ResultsWe showed that PAMAM-dox-glc conjugate exhibits pH-dependent drug release and increased cytotoxic activity compared to free drug in cells cultured in medium without glucose. Further, we proved that these cells overexpress transporters of GLUT family. The toxic effect of conjugate was eliminated by the application of specific GLUT1 inhibitors.ConclusionOur findings revealed that the glucose moiety plays a crucial role in the recognition of cells with high expression of GLUT receptors. By selectively blocking GLUT1 transporter we showed its importance for the cytotoxic activity of PAMAM-dox-glc conjugate. These results suggest that PAMAM-glucose formulations may constitute an efficient platform for the specific delivery of anticancer drugs to tumor cells overexpressing transporters of GLUT family.

Glut 1 in Cancer Cells and the Inhibitory Action of Resveratrol as A Potential Therapeutic Strategy.

An important hallmark in cancer cells is the increase in glucose uptake. GLUT1 is an important target in cancer treatment because cancer cells upregulate GLUT1, a membrane protein that facilitates the basal uptake of glucose in most cell types, to ensure the flux of sugar into metabolic pathways. The dysregulation of GLUT1 is associated with numerous disorders, including cancer and metabolic diseases. There are natural products emerging as a source for inhibitors of glucose uptake, and resveratrol is a molecule of natural origin with many properties that acts as antioxidant and antiproliferative in malignant cells. In the present review, we discuss how GLUT1 is involved in the general scheme of cancer cell metabolism, the mechanism of glucose transport, and the importance of GLUT1 structure to understand the inhibition process. Then, we review the current state-of-the-art of resveratrol and other natural products as GLUT1 inhibitors, focusing on those directed at treating different types of cancer. Targeting GLUT1 activity is a promising strategy for the development of drugs aimed at treating neoplastic growth.

Abstract 3594: GLUT1 as a metabolic target in glioblastoma therapy

Abstract Glioblastoma (GBM) is an aggressive diffuse glioma with poor prognosis due to lack of sound therapeutic options. As in many aggressive tumors, GBMs create a hypoxic microenvironment and switch to glycolytic metabolism from oxidative phosphorylation in what is known as the Warburg Effect. Here, we hypothesize that strategically inhibiting influx of glucose via targeting GLUT1 may reverse the Warburg Effect and selectively slow tumor progression. Nanostring data suggests an aberrant metabolic clincial profile in our cohort of GBM patients. Datamining studies and immunoblot analysis were conducted on these same human GBM specimens to demonstrate upregulated GLUT1. Mass spectrometric analysis was performed on GLUT1 precipitated from mesenchymal subtype patient-derived xenograft (PDX). Zonula Occludens adhesion studies were carried out to determine GLUT1 inhibition's effects on cell-cell adhesion structures. Finally, ATP, lactate release, and glucose uptake assays were performed on PDX and GLUT1-silenced PDX to demonstrate a normalized metabolic phenotype when GLUT1 is silenced. Our work suggests that GLUT1 overexpression plays a major role in the metabolic profile of mesenchymal subtype GBM, and that targeting its overexpression could prove effective in reversing the Warburg Effect in GBM cells and ultimately improving patient outcomes. Citation Format: Collin M. Labak, Maheedhara R. Guda, Neha Jain, Chase P. Smith, Charles P. Cain, Andrew J. Tsung, Kiran K. Velpula. GLUT1 as a metabolic target in glioblastoma therapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3594.

Phloretin Suppresses Bone Morphogenetic Protein-2-Induced Osteoblastogenesis and Mineralization via Inhibition of Phosphatidylinositol 3-kinases/Akt Pathway.

Phloretin has pleiotropic effects, including glucose transporter (GLUT) inhibition. We previously showed that phloretin promoted adipogenesis of bone marrow stromal cell (BMSC) line ST2 independently of GLUT1 inhibition. This study investigated the effect of phloretin on osteoblastogenesis of ST2 cells and osteoblastic MC3T3-E1 cells. Treatment with 10 to 100 µM phloretin suppressed mineralization and expression of osteoblast differentiation markers, such as alkaline phosphatase (ALP), osteocalcin (OCN), type 1 collagen, runt-related transcription factor 2 (Runx2), and osterix (Osx), while increased adipogenic markers, peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), fatty acid-binding protein 4, and adiponectin. Phloretin also inhibited mineralization and decreased osteoblast differentiation markers of MC3T3-E1 cells. Phloretin suppressed phosphorylation of Akt in ST2 cells. In addition, treatment with a phosphatidylinositol 3-kinase (PI3K)/Akt inhibitor, LY294002, suppressed the mineralization and the expression of osteoblast differentiation markers other than ALP. GLUT1 silencing by siRNA did not affect mineralization, although it decreased the expression of OCN and increased the expression of ALP, Runx2, and Osx. The effects of GLUT1 silencing on osteoblast differentiation markers and mineralization were inconsistent with those of phloretin. Taken together, these findings suggest that phloretin suppressed osteoblastogenesis of ST2 and MC3T3-E1 cells by inhibiting the PI3K/Akt pathway, suggesting that the effects of phloretin may not be associated with glucose uptake inhibition.